JP2005530482A - Dominant negative proteins and their use - Google Patents

Abstract

The present invention relates to novel proteins having TNFSF antagonist activity and nucleic acids encoding these proteins. The invention further relates to the use of the novel protein in the treatment of TNFSF-related disorders.

Description

Detailed Description of the Invention

  This application is filed with U.S.S.N.60 / 345,805 filed on January 4, 2002 and U.S.S.N.60 / 373, filed on April 17, 2002, 453, which claims the benefit of 453 filing date, the entire text of which is hereby incorporated by reference.

FIELD OF THE INVENTION The present invention relates to novel proteins of the Tumor Necrosis Factor Super Family (TNSFSF) having antagonist activity and nucleic acids encoding these proteins. The invention further relates to the use of these novel proteins in the treatment of TNFSF-related diseases such as rheumatoid arthritis, sepsis, and autoimmune conditions including, but not limited to, Crohn's disease, and peripheral nerve injury and demyelinating diseases. .

BACKGROUND OF THE INVENTION Multicellular organisms consist of complex and ordered societies of individual cells that must communicate to maintain and regulate their function. This is accomplished by a complex and highly regulated network of hormones, chemical media, chemokines, and other cytokines that act as ligands for intracellular or extracellular receptors. (Bodmer, JL., Et al., (2002) TIBS, 27, 19-26.). These ligands are often activated by ligand-induced oligomerization or conformational changes (Heldin, CH., (1995) Cell, 80, 213-223). The design of proteins that inhibit intercellular signaling processes is significant because many of these proteins can be useful in the treatment of anemia, cancer, diabetes, inflammation, neurological and growth disorders, and other disease states. There is a lot of interest.

  These TNFSF proteins constitute an important class of cytokines involved in a variety of intracellular and intercellular signaling processes. The prototype (prototype) of this family, tumor necrosis factor alpha (TNFA), was originally found to have an in vivo effect of regressing the tumor, but is a key mediator of inflammation. In recent years, this TNFSF is said to constitute at least 18 unique cytokines that exist in secreted and membrane-bound form. These proteins are important regulators of innate and adaptive immune responses and developmental events. They are synthesized as a group of type 2 membrane proteins and are folded into a conserved β-pleated sheet structure that trimers. Most TNFSF components form homotrimers, but there are some exceptions. Lymphotoxin α can form homotrimers, not β, but two of them can also form active heterotrimers with each other. Similarly, APRIL and BLyS together form both homotrimers and heterotrimers. Many of these TNF family members remain membrane-bound and act as cell contact-mediated regulators, while other members are cleaved from the membrane and release the extracellular domain as a regulator.

  Receptors for TNF family members represent a family of structurally related molecules, including at least 26 receptors and / or receptor decoy molecules. This family member extracellular domain group is composed of multiple repeats of cysteine-rich domains (CRD) and small protein domains containing six conserved cysteines forming three disulfide bonds. . The extracellular domain groups of these receptors are even more diverse, although many of this family member contain death domains that mediate apoptosis and other receptor signaling events. All of these members are capable of inducing apoptosis through interaction with one or more groups of intracellular adapter molecules, which may also contain a death domain. Other signaling receptors in this family signal through interactions with a family of adapter molecules called TRAFs (TNF receptor associated factors). Signaling through the TNFSF receptor group is triggered by the binding of oligomeric (and often trimeric) TNFSF ligands.

  Each three-dimensional structure of the TNFSF members is very similar and consists of a sandwich of two anti-parallel beta-sheets with a “jelly roll” or Greek key topology. In addition, all characterized members of this family are assembled into a trimeric complex group. The homologous receptor group of the TNF family ligand group forms a superfamily of related receptor groups. Furthermore, it becomes clear that there is a significant conservation of the receptor binding mode. In general, each receptor monomer binds within a cleft formed between two ligand monomers. It is well documented that inhibition of a set of ligand-receptor systems that both the ligands and the complexes with their receptors are totally similar in three- and four-dimensional structures. This suggests that the strategy developed can also be applied to other proteins in the family. However, no mechanism has been defined so far for the extension of the family to other members. Therefore, the present invention provides a group of methods for creating variants of each member of TNFSF that are antagonistic.

  There still exists a need for proteins that can interfere with intracellular signaling processes. Accordingly, one object of the present invention is to provide a number of TNF superfamilies where each domain has been modified such that its affinity for homologous receptor (s) and / or signaling ability is significantly reduced. It is to provide a protein comprising a receptor-interaction domain. Such linking domains are preferably those that retain association with individual monomeric domains, but are associated with the group of naturally occurring domains associated by segregating in their inactive oligomeric complexes. It shows a dominant negative phenotype that antagonizes the action.

SUMMARY OF THE INVENTION The present invention provides a variant group of extracellular domains of the TNFSF protein group that antagonizes the action of the naturally occurring TNFSF protein group in accordance with the purposes outlined above.

  The present invention provides a group of mutant TNFSF proteins comprising an amino acid sequence having at least one modification relative to the sequence of a naturally occurring TNFSF protein. However, the mutant TNFSF protein group will form a mixed oligomer group that physically interacts with the naturally-occurring TNFSF protein and has substantially no ability to activate receptor signaling.

In another embodiment, the mutant TNFSF proteins of the invention comprise an amino acid sequence that is at least in monomeric form and has at least one modification relative to the sequence of a naturally occurring TNFSF protein, , The mutant TNFSF proteins will interact with the receptor interface at at least one receptor binding site, rendering the receptor substantially incapable of activating receptor signaling Is.
The mutant TNFSF protein group of the present invention preferably has a reduced affinity for the desired receptor as compared with the corresponding wild-type TNFSF protein, and has the ability to interact with other receptor interaction domains. Possesses at least one receptor contact domain.

More specifically, the mutant TNFSF proteins of the present invention physically interact with a naturally occurring TNFSF protein to reduce the ability to activate at least one receptor of the naturally occurring TNFSF protein. The mutant TNFSF proteins of the present invention can interact with corresponding naturally occurring TNFSF proteins or non-corresponding naturally occurring TNFSF proteins.
More specifically, the mutant TNFSF protein group includes at least one modified receptor contact domain with reduced affinity and / or signaling ability for a desired receptor, wherein the protein is desired Is unable to substantially activate its receptor, but retains the ability to interact with other TNFSF proteins.

  In another aspect, the invention contacts a naturally occurring TNFSF monomer protein with a mutant TNFSF monomer protein comprising at least one mutant extracellular domain of the TNFSF protein to form a mixed TNFSF oligomer. A method for antagonizing naturally occurring TNFSF protein is provided. In some cases, for example, the mixed oligomer interacts with the receptor interface at at least one receptor binding site so that the receptor is substantially incapable of signaling activation of the receptor. In some cases, receptor signaling of these mixed oligomers is low compared to wild-type oligomers. In other words or in addition, these mixed oligomers are substantially incapable of activating receptor signaling.

  The present invention relates to the use of a variant group of TNF superfamily protein ligands for the prevention or treatment of various diseases. These variants retain their ability to function as their oligomeric species either in a complex with themselves or with a naturally occurring member of this superfamily, while retaining their ability to biochemical signaling It is specifically engineered to remove or reduce.

  In a preferred embodiment, the mutant TNFSF protein is passed through the receptor as opposed to the wild type TNFSFNFSF protein while maintaining affinity for other TNFSF proteins to form mixed oligomers, most preferably trimers. The technology is designed to produce significantly reduced signal transduction. Such mutant TNFSF protein is referred to as “dominant negative TNFSF mutant” or “DN-TNFSF”. These dominant negative TNFSF variants act by sequestering one or more naturally occurring TNFSF proteins in a mixed heterotrimeric group that is not capable of activating biochemical signaling to an appreciable extent. To do. Thus, the DN-TNFSF protein acts to antagonize the action of naturally produced TNFSF.

  The present invention provides a non-naturally occurring mutant TNFSF protein group (for example, a protein group not found in nature) comprising an amino acid sequence having at least one modification compared to the wild-type TNFSF protein group. . Examples of suitable proteins include, but are not limited to, TNF-α, lymphotoxin-α, lymphotoxin-β, Fas ligand (FasL), TRAIL, CD40 ligand (CD40L), CD30 ligand, CD27 ligand, Ox40 ligand, APRIL , BLyS, 4-IBBL, TRANCE and RANKL (OPGL), and other proteins recognized as being other TNFSF members.

  A preferred embodiment utilizes mutant TNFSF proteins, which interact with one or more of the wild type TNFSF members to substantially activate receptor signaling. It forms a mixed trimer without the ability to convert. Preferably, a mutant TNFSF protein group with at least one amino acid change is used in contrast to the wild type TNFSF protein.

  In other preferred embodiments, each modification can be made either individually or in combination (any combination is possible). In a preferred embodiment, at least one and preferably more positions are utilized in each mutant TNFSF protein. For example, amino acid substitutions are combined to form a double mutant or triple point mutant.

In a further embodiment, one TNFSF molecule can be chemically modified, for example, by PEGylation or glycosylation.
In other embodiments, the N- or C-terminal portion is deleted. In a further embodiment, one TNFSF molecule can be circularly permuted.

In another embodiment, two or more receptor interaction domains of the mutant protein are covalently linked by a linker peptide or other means. Preferably, the linker peptide is a sequence of at least one and not more than about 30 amino acid residues, and includes one or more of the following amino acid residues: Gly, Ser, Ala, or Thr.
In a further aspect, the present invention provides recombinant nucleic acids, expression vectors, host cells and the like encoding non-naturally produced mutant TNFSF proteins.

In another aspect, the present invention provides a method for producing a non-naturally produced mutant TNFSF protein comprising culturing the host cell of the present invention under conditions suitable for expression of the nucleic acid.
In a further aspect, the present invention provides a pharmaceutical composition comprising a mutant TNFSF protein of the present invention and a pharmaceutical carrier.
In a further aspect, the present invention provides a method for treating a TNFSF-related disease comprising administering to a patient a mutant TNFSF protein of the present invention.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the overall mechanism by which a dominant negative TNFSF protein can thereby antagonize the action of a naturally produced TNFSF protein. Each egg type represents a TNFSF protein monomer and each triangle represents a receptor molecule. Naturally occurring mutant TNFSF typically signals by organizing the receptor group into one active complex. Mutant TNFSF proteins have been modified in their receptor contact domains to reduce receptor binding and / or signaling (lumps and sticks). When mutated TNFSF trimers are cultured with naturally produced TNFSF protein trimers, they form and equilibrate to four different trimers, three of which are inactive. A sufficient concentration of dominant negative mutant TNFSF will sequester essentially all naturally produced TNFSF protein into an inactive heterotrimeric complex. Through this and related TNFSF protein inactivation mechanism, the dominant negative TNFSF variants exert their therapeutic effects.

  FIG. 2 shows the structural overlap of TNFSF ligand monomers. Experimentally determined CD40L (1ALY), RANKL (1JTZ), TNFB (1TNR), and TRAIL (1DG6) structures are shown superimposed on the structure of TNFA (1TNF).

  FIG. 3 shows the multiple sequence alignment (MSA) of the human TNFSF member group. FIG. 3 also shows the position numbering of each individual sequence. The numbering for TNF-α (TNFA) and TNFB (LT-α) is based on the current Convention. The numbering for all other sequences is based on the full-length precursor sequence of the protein. For sequences where the ligand-receptor complex structure has been experimentally determined, the residues at the ligand-receptor interface are highlighted in gray. The intermediate surface highlighted in black is used to define the seven total receptor contact regions of the TNF superfamily ligands. A general numbering system starting at position number 1 is also included at the top of the MSA for reference.

  FIG. 4A is a chart showing that the TNF-alpha variant group has been pre-exchanged with wild-type TNF-alpha to reduce TNF-alpha-induced NFkB activation in 293T cells. FIG. 4B shows that exchange of wild-type TNF-alpha with A145 / Y87HTNFSF mutants inhibits TNFSF-induced nuclear translocation of NFkB in HeLa cells. FIG. 3 is an immuno-localization photograph of NFkB. FIG. 4C shows that TNFSF variant A145R / Y87H reduces wild type TNFSF-induced activation of the NFkB-induced luciferase reporter.

FIG. 5 is a chart showing the antagonist activity of the TNF-alpha variant.
6A-C are dose response curves for caspase activation by various TNF-alpha variants.

  FIG. 7A shows a dominant-negative inhibitory RANKL mutant strategy model. RANKL mutant homotrimers and heterotrimers with wild type RANKL do not activate the RANK receptor. RANKL variants act in a dominant-negative manner that inactivates wild-type RANKL protein through the formation of these heterotrimeric species. The knobs and sticks on the designed RANKL protein symbolize the theoretically designed mutations that interfere with receptor binding in the small and large binding domains of RANKL. Via this mechanism of inactivating wild-type RANKL, the dominant-negative inhibitory RANKL mutant groups of the present invention exert their therapeutic effects.

FIG. 7B shows a list of computer designed inhibitory RANKL variants. These inhibitory variants can be combined with the RANKL soluble variant to produce a soluble human RANKL variant.
FIG. 7C shows that RANKL soluble mutant C22S / I247E-mediated osteoclast development is antagonized by OPG and RANK-Fc. Each plot shows that the RANKL product from BioSource is antagonized to the same extent as the soluble variant from Xencor. The BSA and Enbrel controls indicate that this antagonism is specific for RANKL that interacts with OPG and RANK-Fc. RANK-Fc and OPG controls show that they do not mediate osteoclast development.

  FIG. 7D shows the RANKL mutant antagonist screen. The ability of the RANKL mutant to antagonize osteoclastogenesis was assessed by monitoring TRAP levels when RANKL-C221S / I247E was mixed with the RANKL mutant, cultured, and added to RAW264.7 cells. TRAP is released from RAW264.7 cells as RANK-mediated osteoclast development proceeds. This experiment identified a RANKL variant that antagonized this process. FIG. 7E. RANKL mutant antagonism screen using a fixed ratio of RANKL-C221S / I247E to a 10-fold excess mutant. The ability of RANKL mutants to antagonize osteoclastogenesis is to monitor TRAP levels when RANKL-C221S / I247E is mixed with RANKL mutant, cultured, diluted to 2 logs or more and added to RAW264.7 cells. Evaluated by. TRAP is released from RAW264.7 cells as RANK-mediated osteoclast development proceeds. This experiment identified a RANKL variant that antagonized this process. FIG. 7F shows the RANKL mutant antagonist screen. The ability of RANKL mutants to antagonize osteoclastogenesis was assessed by monitoring TRAP levels when RANKL-C221S / I247E and RANKL mutants were added to RAW264.7 cells but not precultured. TRAP is released from RAW264.7 cells as RANK-mediated osteoclast development proceeds. This experiment identified a RANKL variant that antagonized this process. FIG. 7G shows a summary of RANKL antagonistic variants. These variants have been shown to antagonize RANKL-mediated osteoclast development.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel class of proteins that exhibit a dominant-negative phenotype or mechanism of action. The dominant-negative therapeutic strategy of the present invention is a novel TNFSF variant with reduced receptor binding and / or activation properties and the ability to oligomerize a naturally produced TNFSF protein compared to a naturally produced TNFSF protein. (Fig. 1). In other words, the TNFSF variant group that leads to a decrease (preferably substantially no activation) of the TNFSF receptor group (compared to the naturally occurring TNFSF protein) is exchanged for at least one naturally occurring TNFSF protein. It is sequestered in a reduced or inactivated hetero-oligomer and inhibits the biological activity of those oligomers, eg, the ability to bind and / or activate the receptor.

The dominant-negative TNFSF variants of the present invention can be designed by modifying the TNFSF protein at the key receptor contact point to disrupt the ability of either ligand to bind or activate the receptor. Exchange and physical interaction of these oligomeric TNFSF variants with naturally produced TNFSF proteins results in reduced activity or deactivation of the naturally produced TNFSF protein. In order to more effectively achieve this goal, the TNFSF variants of the present invention can also be designed to preferentially hetero-oligomerize naturally produced TNFSF proteins. Alternatively, these variants bind to each other and depend on the amount of variant oligomers present; for example, the equilibrium of the variant mixed oligomers favors the exchange of any naturally produced TNFSF protein. It can also be designed to go away.
Accordingly, the present invention provides methods and compositions that utilize mutants of the extracellular domain of TNFSF protein that antagonize naturally occurring TNFSF protein.

  “Extracellular domain” or “ECD” as used herein means a segment that predominates in the extracellular portion of a protein, which is generally soluble when separated or isolated from the rest of the protein. is there. For transmembrane proteins, this segment can be tethered to the cell via a transmembrane domain or released from the cell by proteolytic digestion. Alternatively, the extracellular domain may comprise the entire protein or amino acid segments thereof when secreted from the cell. In general, TNFSF members are expressed as a group of II transmembrane proteins (extracellular C-terminus). This unprocessed protein generally contains an atypical signal anchor / intracellular domain of about 10 to 80 amino acids. The extracellular region can be about 140-215 amino acids long. Soluble forms of the TNFSF protein are obtained by proteolytic cleavage of the signal propeptide by matrix metalloproteinases called TNF-alpha converting enzymes (TACE) or by direct recombinant methods. FIG. 3 shows a number of extracellular domains from a number of different TNFSF proteins. As will be appreciated by those skilled in the art, these domains can be shorter or longer than those shown in FIG.

In a preferred embodiment, this extracellular domain can be functionally defined as a TNFSF protein or mutant protein that is soluble and forms oligomers (preferably with wild type monomers).
Unless otherwise disclosed, the mutant TNFSF proteins of the invention are composed of extracellular domains or functional equivalents thereof. That is, the mutants of the present invention do not contain a transmembrane domain unless otherwise specified. In one embodiment of the invention, the mutant TNFSF protein of the invention antagonizes the membrane-bound native production form of the TNFSF protein, and in other embodiments, the mutant protein of the invention is soluble in the naturally produced TNFSF protein. It antagonizes the form or both.

  The TNFSF protein of the present invention is a mutant protein. The mutant TNFSF proteins and nucleic acids of the invention are distinguished from naturally occurring or wild type TNFSF. “Naturally produced”, “wild type”, “natural” or grammatically equivalent phrase means an amino acid sequence or nucleic acid found in nature and includes allelic changes; ie, amino acid sequences that are not normally deliberately modified Or a nucleic acid sequence. Thus, “non-naturally-occurring” or “synthetic” or “recombinant” or grammatically equivalent phrases herein refer to amino acid or nucleic acid sequences that are not found in nature; Amino acid sequence or nucleic acid sequence. As used herein, the term “recombinant nucleic acid” generally refers to a nucleic acid that is uniquely formed in vitro in a form that is not normally found in nature by manipulating the nucleic acid with an endonuclease. Thus, both isolated linear mutant TNFSF nucleic acids or expression vectors formed in vitro by ligation to normally unbound DNA molecules are considered recombinant for the purposes of this invention. It is done. It is understood that once a recombinant nucleic acid has been created and reintroduced into a host cell or organism, it will replicate non-recombinant, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulation. However, once such nucleic acid is produced recombinantly, it will still be considered recombinant for the purposes of the present invention, even if replicated non-recombinantly thereafter.

  Similarly, a “recombinant protein” is a protein made using recombinant techniques, ie, by expression of a recombinant nucleic acid as described above. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein can be isolated and purified from some or all of the proteins and compounds normally associated with it in its wild-type host, and can thus be made substantially pure. For example, an isolated protein, in its native form, is usually accompanied by at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample. It is not accompanied by at least some of the ingredients. The substantially pure protein constitutes at least about 75%, preferably at least about 80%, particularly preferably at least about 90% of the total protein weight. This definition includes the production of mutant TNFSF proteins from one organism in a different organism or host cell. Alternatively, the protein can be produced at concentrations significantly higher than those normally found by the use of inducible promoters or high expression promoters, thus making the protein at increased concentration levels. In addition, all of the mutant TNFSF proteins of the invention outlined herein are amino acid substitutions, insertions and deletions, preferably substitutions, as compared to the corresponding wild-type endogenous sequences. In the form not normally found in nature.

  The amino acid sequence of a representative naturally occurring human TNFSF is shown in FIG. It should be noted that the position numbers of mutant TNFSF proteins and mutant TNFSF nucleic acids are all based on these sequences, unless otherwise noted. That is, as will be apparent to those skilled in the art, the alignment of the TNFSF protein and the mutant TNFSF protein can be made by identifying an “equivalent” position between the two proteins using a standard program, as outlined below. . Accordingly, the mutant TNFSF proteins and nucleic acids of the present invention are non-naturally occurring; i.e., non-naturally occurring.

  In a preferred embodiment, the mutant TNFSF protein of the invention contains non-conservative modifications (eg, substitutions). As used herein, a “non-conservative” modification is a significant difference between wild-type and mutated residues in one or more physical properties, including hydrophobicity, charge, size and shape. Means the modification. For example, polar residues to nonpolar residues or vice versa, positively charged residues to negatively charged residues, or vice versa, and large residues to small residues or vice versa The reverse modification is a non-conservative modification. For example, substitutions may be made that more significantly affect the structure of the polypeptide backbone in the altered region, such as alpha-helical or beta-sheet structures; molecular charge or hydrophobicity at the target site; or side chain bulk: obtain. In general, the substitutions that are expected to produce the greatest changes in polypeptide properties are as follows: (a) a hydrophilic residue such as seryl or threonyl is a hydrophobic residue such as leucyl, Substituted by (or by) isoleucyl, phenylalanyl, valyl or alanyl; (b) cysteine or proline is substituted by (or by) some other residue; (c) electropositive A residue having an unfavorable side chain, such as lysyl, arginyl, or histidyl, is substituted (or by) a residue having an electronegative side chain, such as glutamyl or aspartyl; or (d) bulk Residues with high side chains, eg phenylalanine, without side chains, eg substituted with (or by) glycine Etc. it is. In a preferred embodiment, the mutant TNFSF protein of the invention has at least one non-conservative modification.

Conservative modifications are generally as shown below. However, as is known in the art, there can be other substitutions that are considered conservative:

  Protein modifications are preferably substitutions, which include those on the surface, boundaries and core regions of TNFSF members. See, for example, US Pat. Nos. 6,188,965 and 6,269,312 (which are incorporated herein by reference). In another preferred embodiment, modifications can be made to surface residues, particularly if alteration of binding properties (to any other monomer or receptor) is desired.

Mutant proteins have been previously described, for example, in U.S. Patent Nos. 6,188,965; 6,296,312; 6,403,312; U.S.S.N.s09 / 419,351,09. / 782,004,09 / 927,790,09 / 877,695, and 09 / 877,695 ^{PDA TM} systems are described in; alanine scanning method (see U.S. Pat. No. 5,506,107), gene shuffling Use gene shuffling (WO01 / 25277), site saturation mutagenesis, mean field, sequence homology, or other methods known to those skilled in the art to guide the selection of point mutation sites and types Can be generated.

  In preferred embodiments, sequence and / or structural alignments can be used to generate mutant proteins of the invention. Numerous as known in the art; for example, Smith-Waterman search, Needleman-Wunsch, Double Affine Smith-Waterman frame search, Gribskov / GCG profile search, Gribskov / GCG profile scan, profile There are sequence-based alignment programs including Frame Search, Bucher Generalization Profile, Hidden Markov Model, Hframe, Double Frame, Blast, Psi-Blast, Clustal, and GeneWise. A wide variety of structural alignment programs are known. For example, VAST derived from NCBI (http://www.ncbi.nlm.nih.gov:80/Structure/VAST/vast.shtml); SSAP (Orengo and Taylor, Methods Enzymol 266 (617-635 (1996)) SARF2 (Alexandrov, Protein Eng 9 (9): 727-732. (1996)) CE (Shindyalov and Bourne, Protein Eng 11 (9): 739-747. (1998)); (Orengo et al., Structure 5 (8 ): 1093-108 (1997); Dali (Holm et al., Nucleic Acid Res. 26 (1): 316-9 (1998)), all of which are incorporated herein by reference.

  The methods of the invention can be applied to any certified member of TNFSF or related (eg, c1q family of proteins) systems in which individual domains oligomerize to form active complexes. These domains can be modified in a number of ways to delete or reduce receptor binding and / or activation. In addition, each modification domain can be covalently coupled with at least one other modification domain to generate a group of dominant negative proteins with enhanced antagonistic activity.

  In a preferred embodiment, these proteins are TNSFSF (Oren, DA, et al., (2002) Nature Structural Biology, 9, 288-292; Bodmer, JL., Et al., (2002) TIBS, 27, 19- 26; Locksley, RM, et al., (2001) Cell, 104, 487-501; see WO 01/25277; all of which are hereby expressly incorporated by reference. Thus, the definition of “TNFSF” protein includes, but is not limited to, a TNFSF member of interest. TNFSF members of interest include ligands for TNF; osteoprotegerin (OPG), also known as RANKL (US 5,843,678, incorporated herein by reference), CD40 ligand , BLyS, etc. and others shown in FIG. However, in some embodiments, the TNFSF specifically excludes the TNF-α protein.

  The compositions and methods of the present invention include, for example, structural homologs of TNFSF, including the C1q-related family of proteins, such as the 30 kDa adipocyte complement-related protein (ACRP30) and its human -Applicable to include ortholog APM-1. ACRP30 belongs to the C1q-related family of proteins and contains at least 23 members, either with or without collagen domains. Those that contain collagenous domains include ACRP30, C1qA, B, and C chains, hibernation in the cabbage-related proteins (HP-20, 25, and 27), CORS26, and the elastin microfibril interface- There is a localized protein (EMILIN). Those lacking the collagen domain include multimerin and the precursors of cerebellins 1 and 3. Many of these proteins, which also form trimers or multimeric trimers, have been implicated in development as well as in immunological and physical homeostasis.

  As outlined herein, mutant TNFSF proteins serve as antagonists to wild-type proteins by a variety of mechanisms. In a preferred embodiment, the mutant protein group of the present invention forms a hetero-oligomer group with the endogenous wild type TNFSF protein group, and then binds (but does not activate) the corresponding receptor group. That is, as illustrated in FIG. 1, the mutant TNFSF protein of the present invention is preferably modified to disrupt the interaction with the receptor molecule. Preferably, these modifications are those that do not substantially affect the ability of the mutated domain to interact with and sequester the naturally occurring TNFSF protein. In preferred embodiments, these modifications can be combined with another modification that enhances the ability of the mutant TNFSF proteins to hetero-oligomerize with one or more naturally occurring TNFSF proteins. Particularly preferably, modifications that affect receptor activation and oligomerization are also combined with chemical modifications (eg, glycosylation, PEGylation, fusion, etc.) that improve pharmacokinetic properties, as further outlined below.

  More preferably, the present invention also relates to novel proteins and nucleic acids having TNFSF antagonist activity. Thus, a mutant protein having “antagonist” activity will ultimately lack receptor activation, as further defined below. This is because mutant monomers interact with wild-type monomers and “antagonize” wild-type monomers, for example, so that wild-type monomers can interact with receptors. By the lack of the ability to bind and / or activate. Overall, these mixed oligomers contain at least one mutated monomer and at least one wild type (eg, endogenous) monomer inhibits receptor activation.

  In a preferred embodiment, the mutant TNFSF protein group of the present invention is a dominant negative protein. A “dominant negative” phenotype or “mechanism of action” is used herein to have reduced affinity for at least one desired receptor and / or altered signaling, and as such A mutant TNFSF single molecule that retains the ability of the protein to oligomerize with other receptor interaction domains (but the monomer is substantially incapable of interacting and / or signaling with the desired receptor) It means a protein containing a monomer (see FIG. 1). Depending on the composition of the oligomeric ligand complex, i.e., 2 variants for heterotrimers: 1 native, or 1 variant: 2 native, the degree of inhibition of ligand-mediated receptor activation is altered (Fig. 1). In other words, receptor activation is completely inhibited in complexes containing 2 variants: 1 natural type, but decreased in complexes containing other ratios of variants: natural type. Alternatively, it can be said that receptor activation is not completely inhibited and decreases to a degree that varies based on the composition of the heterodimer and the corresponding mode of action. See also Menart, V., et al., (2000) Eur J Physiol., 439, R113-R115; US Patent Pub. Nos. 2002/0039588, 2002/0040132, 2002/0037286, 2002/0037280; all of them Reference is made to this specification. Monte carlo simulation of heterotrimeric assembly as a function of the relative concentration of mutant TNFSF to naturally produced TNFSF generally results in natural TNFSF when a 10-fold excess of mutant TNFSF monomer is added. It shows that 99% or more of the monomers were sequestered.

  As will be appreciated by those skilled in the art, two main approaches for creating the dominant negative variants of the present invention include: (1) modifying individual receptor interaction domains to allow receptor binding and / or Reducing or deleting signaling; and (2) covalently coupling modified receptor interaction domains to enhance inhibition of receptor activation.

  In preferred embodiments, individual TNFSF protein groups are modified within their receptor contact domains to reduce or eliminate receptor binding and / or signaling. For example, amino acid substitutions can occur as modifications that reduce or eliminate receptor binding in the receptor contact domain. In preferred embodiments, at least one non-conservative variant in the receptor contact domains can be created to disrupt receptor interaction. Figures 4A and 4B, and 5,506,107; U.S.S.N.s09 / 798,789; 09 / 981,289; 10 / 262,630; 60 / 374,035; and the name "NOVEL VARIANTS OF RANKL PROTEIN ", filed on the same day, all of which are hereby incorporated by reference.

  Preferred modifications that affect receptor binding and / or signal transduction (eg, substitutions, insertions, deletions, etc.) are confirmed using a variety of techniques, including structural alignment methods, sequence alignment methods, etc. as described above. can do. In many cases, the group of amino acids that interact with the receptor in the TNFSF ligand can be identified directly from the three-dimensional structure of the TNFSF ligand-receptor complex. Alternatively, equivalent information can be derived by analysis of ligand-receptor complexes of related proteins. For example, within this TNFSF, TNF family members can be structurally aligned with certain TNFSF proteins whose structure has been experimentally determined (eg, Oren, DA, et al., (2002). ) Nature Structural Biology, 9, 288-292). Alternatively, sequence alignment can be used if it is not possible to structurally align residues.

  As is known in the art, there are a number of sequence alignment methodologies that can be used. For example, alignment methods based on sequence homology can be used to create a sequence alignment of each TNFSF member (Altschul et al., J. Mol. Biol. 215 (3): 403-410 (1990); Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997), both of which are incorporated by reference).

  In a preferred embodiment, as highlighted in FIG. 3, the amino acid sequences of each member of the TNF superfamily can be aligned in a multiple sequence alignment (MSA). The alignment shown in FIG. 3 is derived initially from the Pfam database and then further utilizes the structural alignment (using CE) of the TNFA, TNFB, CD40L, TRAIL, and BlyS crystal structures. This MSA can also be used for each other recognized TNFSF member and other structural homologues and families, expanding the structural known information about them. Due to the high degree of structural homology between different TNFSF members, this MSA can be used as a reliable predictor of the effects of modifications at various positions within the alignment. Thus, the TNFA sequence and numbering shown in FIG. 3 can be used as an MSA reference point for all other TNFSF protein sequences. As used herein, reference to “position of TNFSF protein corresponding to TNFA amino acid X” means reference to a collection of other recognized TNFSF members and other structural homologues and equivalent positions in the family. . For example, each position of the TNFSF protein corresponding to the TNFA amino acid L75 is the following amino acid position in the following TNFSF protein: TNFA: L75, TNFB: Y96, FASL: P206, LIGHT: T161, VEGI: S99, lymphotoxin beta : T158, APRIL: T177, BLyS: A207, CD40L: P188, RANKL: Q237, TRAIL: Q205, CD27L: S121, 4-1BBL: S162, TWEAK: Y176, CD30L: D157, OX40L: N114, AITRL: N106, and , Corresponding to each equivalent position in other proteins recognized as TNFSF members.

  For example, structural analysis of a complex of TNFB and the p55 (R1) receptor indicates that amino acid Y108 in TNFB is in direct contact with the receptor. A homologous residue Y216 from TRAIL is also in direct contact with the DR5 receptor. The MSA thus predicts that the TNFA-derived homologous residue I97 is also in direct contact with the receptor. Consistent with this prediction, mutation of TNFA-I97 to R or T results in a significant loss of receptor-binding affinity and biological signaling activity. The analysis for this contact position can be extended to all members of this family, predicting that the following positions are important for receptor interaction: FASL: Y218, LIGHT: Y173, VEGI: Y111, TNFC: Y170, APRIL: R189, BlyS: V219, CD40L: R200, RANKL: I249, CD27L: C133, 4-1BBL: A174, TWEAK: A188, CD30L: K169, OX40L: L126, and AITRL: Y118. This type of analysis can be performed on all receptor contact regions of these ligands.

  FIG. 3 highlights the 7 canonical receptor contact area based on analysis of known structure and mutation data. In a preferred embodiment of the invention, each of the seven regions highlighted in FIG. 3 as receptor contact regions is used to determine each modification site for creation of a variant of each TNFSF member. In another preferred embodiment, such modifications reduce receptor affinity and / or signaling ability. In another preferred embodiment, these modifications also preserve the ability of each protein to oligomerize with the naturally occurring TNFSF protein, including but not necessarily limited to the corresponding wild type sequence of each family member.

  If the alignment system shown in FIG. 3 or any other alignment program described above is used, the numbering system of all alignment programs can be used as a reference point, and the matching location of the TNFA protein can be It can be correlated with members or structural homologues recognized as TNFSF members as well as equivalent positions in the family.

  For the purposes of the present invention, these regions of the TNFSF protein to be modified are preferably selected from the group consisting of (but not necessarily) a large domain, a small domain, a DE loop, and a trimer interface. Is done. These large, small, and DE loops are three separate receptor contact domains, each from several non-contact linear segments of the protein (ie, the seven canonical receptor contact regions described above). It is made up. These domains are lymphotoxin-alpha and TRAIL, two TNFSF proteins whose structures (PDB entries 1TNR and 1D0G, respectively) have been determined in complex with their cognate receptor group, Are identified in TNFSF protein- and MSA- by contrasting with the respective receptor interaction domains using crystallography. This trimer interface mediates interactions between individual TNFSF protein monomers. The trimerization position can be either directly from the crystal structure of the appropriate TNFSF protein (eg, for TNFA, TNFB, BLyS, TRAIL, CD40L, or RANKL) or homologous to the other TNFSF protein. Can be identified. In a preferred embodiment, a position group containing an atomic group derived from a certain TNFSF protein monomer and having a distance within 5 angstroms from an adjacent monomer is defined as a trimer intermediate plane position. The modification is performed in one of these regions alone or in any combination with other regions.

  Preferred positions for large domains that modify in the TNFSF protein include, but are not limited to, the corresponding 28-34, 63-69, 112-115, and 137-147 positions of TNFA. For small domains, preferred positions to be modified include, but are not limited to, the corresponding 72-79 and 95-98 positions of TNFA. For the DE loop, preferred positions to be modified include, but are not limited to, the corresponding 84-89 positions of TNFA. These trimer intermediate plane positions to be modified include TNFA corresponding 11, 13, 15, 34, 36, 53-55, 57, 59, 61, 63, 72, 73, 75, 77, 119. , 87, 91-99, 102-104, 109, 112-125, 147-149, 151, and 155-157 position groups, but are not limited thereto. Particularly preferred trimer interface positions to be modified are the corresponding 57, 34, and 91 positions of TNFA. For example, amino acids X and Y at the corresponding 34 and 91 positions of TNFA may be replaced simultaneously with a similarly charged group of amino acids (eg, X34E + Y91E, X34K + Y91R, etc.) to disrupt the stability of the mutant-natural interface. In addition, electrostatic repulsion can be generated at the mutated monomer-monomer intermediate surface.

  In preferred embodiments, the site and type of modification is selected with reference to other sequences of the alignment. Thus, in a preferred embodiment, the initial amino acid X from sequence A is mutated to amino acid Y from sequence B, thus making Y a non-conservative substitution in relation to amino acid X. For example, amino acid Y87 from TNFA is aligned with non-conserved R189 from APRIL. In fact, as shown in previous studies, the Y87R substitution in TNFA leads to a significant decrease in receptor binding and signaling by TNFA. In another embodiment, more conservative mutations can also be utilized. In another embodiment, the wild type residue is mutated to alanine.

In a preferred embodiment, useful modifications at the receptor contact and / or trimerization interface are protein design or PDA ^TM techniques (US 6,188,965; 6,269,312; 6,403,312; USSN 09). 09 / 812,034; 09 / 827,960; 09 / 837,886; 09 / 782,004 and 10 / 218,102, all of which are incorporated herein by reference) Selected using algorithmic modeling. As is known in the art, this class of algorithms generally uses atom-level or amino acid-level scoring functions to determine the overall tertiary and quaternary of a protein. Evaluate amino acid sequence compatibility with structure. Thus, this class of algorithms is used to disrupt receptor-binding, which does not substantially disrupt the ability of the mutant TNFSF protein to properly fold and oligomerize with itself or with their naturally produced target). Can be selected. These techniques typically use high-resolution structural information input of the target protein. In a preferred embodiment, the experimentally determined structure of the appropriate TNFSF protein is used as input. In another embodiment, using MSA, an atom-level homology model for TNFSF members (based on a subset of families whose three-dimensional structure has been determined using crystallography or related methods). Can be guided. For TNFSF, high-resolution structures have been determined for TNFA, TNFB (LT-alpha), TRAIL, CD40L, and BLyS. These homology models can be used alternatively as structural scaffolds to guide the design of mutant TNF superfamily ligands with reduced receptor binding and / or signaling and / or dominant-negative activity. Can do.

  In another embodiment, protein design algorithms can be used to generate mutations in individual receptor interaction domains that create steric repulsion between the receptor interaction domain and the receptor. Other mutations that can occur include, but are not limited to, mutations that create electrostatic repulsion and mutations that create desolvation of adverse amino acid groups.

  In preferred embodiments, multiple receptor interactions and / or substitutions, insertions, deletions or other modifications in the trimerization domain may be combined. Their combination is often advantageous in that they have an additive or synergistic effect on the dominant-negative activity. Examples include large and / or small domains (eg, MSA positions 94 and 112), large domains and DE loops (eg, MSA positions 95 and 124), large domains and trimerization domains (eg, These include, but are not limited to, simultaneous substitutions of amino acid groups at MSA positions 94 and 83) or multiple substitutions within a single domain. Examples other than these include all combinations of each substitution.

  In preferred embodiments, the defined receptor contact region constitutes a site for insertion, deletion or substitution of amino acid residues, or a site for introduction of a chemically modified site. In a preferred embodiment, the deletion or insertion is performed according to MSA. For example, MSA studies have revealed that BLyS amino acids 220-223 constitute a 4-residue insertion associated with many other family members. This region is present in the DE loop region of proteins that are well known to interact directly with receptors in many TNF ligand-receptor systems. Thus, the deletion of these four residues is expected to reduce the affinity of BLyS for its receptor, while it is predicted that the structural accumulation of BLyS protein on the other hand will be maintained.

  In further embodiments, each of the variants described above can be combined with other modifications to the TNF superfamily ligand. These include, but are not limited to, additional amino acid substitutions, insertions or deletions, and / or post-translational modifications such as chemical (eg, PEGylation) or glycosylation (WO 99/45026; WO01 / 49830; WO01 / 49830; WO02 / 02597; WO01 / 58493; WO01 / 51510, US Patent Nos. 4,002,531; 5,183,550; 5,089,261; 6,153,265; 5,264,209; 5,383,657; 5,766,897; 5,986,068; 4,280,953; 5,089,261; 5990237; 6461802; 6495659; 6448369; 6437025; 5900461; 5446090; 5672662; 5919455; 61139 6: 5985236; 6214966; 6258351; 5932462; EP0786257; EP0905205; EP1064951; EP0545426; EP0424405; EP0400472; As part of). In some embodiments, such as animal models of disease, fusion proteins comprising other sequences of mutant TNFSF proteins can be implemented. For example, labels (eg, autofluorescent proteins, survival and / or selection proteins), stability and / or purification sequences, toxins, mutant proteins from other members of the superfamily (eg, “dual It can be carried out using a fusion partner that includes (similar to the creation of “bi-specific antibody populations”) or other utility protein sequences. Additional fusion partners are described below. In some cases, these fusion partners are not proteins.

  In preferred embodiments, additional amino acid substitutions are made to optimize hetero-oligomer interactions between the mutant ligand and its internal counterpart and / or to destabilize the oligomeric state in the case of the mutant alone. For example, certain TNFA intra-L57F mutations are inconvenient for the formation of mutant homotrimers, but continue to be designed to promote the formation of mutant: natural heterotrimers. Such modifications are useful to facilitate the exchange of mutant monomers with natural monomers and promote a dominant-negative mechanism of action.

  As will be appreciated by those skilled in the art, mutant TNF superfamily ligands with reduced signaling ability can be found by a variety of methods. These methods include directed evolution (eg, error-prone PCR, DNA shuffling, etc.), single-site saturation mutagenesis, and alanine-scan mutagenesis. There is no limitation to them. In addition, the use of these or other methods may result in substitutions, insertions, or deletions that occur outside the seven normative contact regions described herein—which reduce receptor binding and / or signaling activity— Allow discovery.

  In other embodiments, coiled-coil motifs are used to aid dimer assembly (Dahiyat et al., Protein Science 6: 1333-7 (1997) and USS N.09 / 502,984); All of which are incorporated herein by reference in their entirety). The helical-coil motif is based on the structure of GCN4: RMEKLEQKVKELLRKNERLEEEVERVERQQLVGER; AALESKVSALESEVASLESEVAAL, and LAAVKSKLSAVKSKLSKSVSKLAA, as defined above. 1994), which is incorporated herein by reference, but is not limited thereto. For example, other coiled-coil sequences derived from protein-containing leucine zippers are also known in the art and used in the present invention. (See, eg, Myszka et al., Biochem. 33: 2362-2373 (1994), incorporated herein by reference).

Similarly, using molecular dynamics calculations, sequences can be screened computationally by calculating mutant sequence scores individually and creating a list.
In a preferred embodiment, each residue pair potential can be used to score each sequence during computational screening (Miyazawa et al., Macromolecules 18 (3): 534-552 (1985), see, see Expressly incorporated herein).

As will be apparent to those skilled in the art, additional TNFSF proteins can be identified and added to the MSA highlighted in Figure X. The sources of these sequences vary widely and include sequences taken from sequences from one or more known databases [GenBank (http://www.ncbi.nlm.nih.gov/)), including Non-limiting].
In addition, sequences from these databases can be subjected to contact analysis or gene prediction; Wheeler, et al., Nucleic Acids Res 28 (1): 10-14. (2000), and Burge and Karlin, See J Mol Biol 268 (1): 78-94. (1997).

  As is known in the art, there are a number of usable methodologies for sequence alignment. For example, an alignment method based on sequence homology (Altschul et al., J. Mol. Biol. 215 (3): 403-410 (1990); Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997) Can be used to create a sequence alignment of each TNFSF member. These sequence alignments are then examined to determine the observed sequence modifications. These sequence modifications are displayed and the mutant TNFSF protein is determined.

  Sequence-based alignments can be used in a variety of ways. For example, as is known in the art, a large number of related proteins can be aligned to determine “variable” and “conservative” residues; ie, alter identity between the family members Each residue to be retained or retained can be identified. These results can be used to guide the design of mutant protein libraries that can experimentally probe their properties. For example, positions that are highly variable (ie, low conservative) among family members can be randomized using either all amino acids or a subset thereof. Alternatively, sequence modifications can be displayed and appropriate substitution groups can be determined therefrom. Alternatively, accepted sequence modifications can be used to determine the group of amino acids considered at each position during the computational modeling and / or screening process. Another modification is to bias the score for amino acid groups that occur during sequence alignment, thereby increasing the probability found during computational screening but still allow for other amino acid group considerations. . This bias will result in a library of targeted mutant TNFSF proteins, but does not preclude considering amino acids that were not found in the alignment.

As used herein, a mutant TNFSF or TNFSF protein protein includes a TNFSF monomer group, a dimer group, or a trimer group, and includes a wild-type protein in a trimer group. When incorporating, the former two are preferable.
These TNFSF protein groups may be derived from any kind of organism, but those derived from mammals having the TNFSF protein group are particularly preferred. Suitable mammals include rodents (rats, mice, hamsters, guinea pigs, etc.), primates, livestock animals (including sheep, goats, pigs, cows, horses, etc.); (The arrangement of which is shown in FIG. 6B) is included but is not limited thereto. As will be apparent to those skilled in the art, TNFSF proteins based on non-human mammalian TNFSF proteins can be used in animal models of human disease.

  The mutant TNFSF protein of the present invention is an antagonist of a naturally produced TNFSF protein. As used herein, “a naturally occurring TNFSF antagonist” refers to a mutant TNFSF protein that inhibits activation of receptor signaling as opposed to activation by a naturally occurring member of TNFSF in the absence of the antagonist. Or it means to reduce significantly.

  In a preferred embodiment, the mutant TNFSF protein of the present invention physically interacts with and so does its corresponding wild-type TNFSF protein (ie, the endogenous naturally produced TNFSF protein from which the mutant was derived). Thus, the complex containing the mutant TNFSF and the wild-type TNFSF protein is rendered incapable of activating the TNFSF receptor. Preferably, the mutant TNFSF protein of the present invention interacts preferentially with the wild-type TNFSF protein to form a mixed trimer with the wild-type TNFSF protein, so that receptor binding is Prevent it from occurring and / or prevent TNFSF signaling from starting (FIG. 1).

  In another embodiment, the mutant TNFSF protein of the invention physically interacts with a non-corresponding wild-type protein (ie, a naturally occurring protein that is different from the protein from which the mutant was derived). For example, dominant-negative mutant APRIL proteins are known to form homotrimers on their own, and to form heterotrimers with BLyS, so that the dominant-negative mutant APRIL protein is a naturally occurring APRIL or BLyS. Can be used for protein inhibition. Similarly, since lymphotoxins α and β form an active heterotrimer with each other, the dominant-negative mutant LT-α protein of the present invention can be used to inactivate LT-β. it can.

  As used herein, mixed trimer means that monomers of naturally occurring and mutant TNFSF proteins interact to form trimer TNFSF (FIG. 1). The mixed trimer may comprise 1 mutant TNFSF protein: 2 natural TNFSF protein, 2 mutant TNFSF protein: 1 natural TNFSF protein. In some embodiments, a trimer can be formed comprising only the mutant TNFSF protein (FIG. 1B).

  In a preferred embodiment, the mutant TNFSF antagonist protein of the invention is an antagonist that is highly specific for the corresponding wild-type TNFSF protein. However, in another embodiment, the mutant TNFSF antagonist protein of the invention is specific for one or more wild-type TNFSF proteins. For example, mutant APRIL proteins may be antagonists specific for wild-type APRIL protein only, wild-type APRIL and BLyS, and wild-type BLyS only. Additional features of the mutant TNFSF antagonist proteins of the present invention include improved stability, pharmacokinetics, and a high affinity for naturally occurring TNFSF. Variants with a high affinity for naturally occurring TNFSF can be generated from variants that exhibit TNFSF antagonism as described above.

  In a preferred embodiment, the mutant TNFSF protein exhibits a decreased biological activity compared to native TNFSF. This includes, but is not limited to, decreased receptor binding, decreased activation and / or eventual loss of other unwanted activity that can lead to cytotoxic activity or adverse side effects. As used herein, “cytotoxic activity” means the activity of a TNFSF variant that selectively kills or inhibits cells. Variant TNFSF proteins that exhibit less than 75-50% biological activity relative to nature are preferred. More preferred are mutant TNFSF proteins that exhibit less than 25% of biological activity of naturally occurring TNFSF, even more preferred are mutant TNFSF proteins that exhibit less than 15%, most preferred 10 It is a mutant TNFSF protein showing less than%. Suitable assays include TNFSF cytotoxicity assays, DNA binding assays; transcription assays (using reporter constructs; see Stavridi, supra); size exclusion chromatography assays and radiolabeling / immunoprecipitation (see Corcoran et al., Supra). ); And stability assays (including but not limited to the use of circular polarization dichroism (CD) assays and equilibrium studies; Mateu, supra). All of which are hereby incorporated by reference. These assays can utilize labeled mutant proteins. Suitable labels include radioisotopes, chromophores (particularly fluorophores), enzymes, particles (eg, magnetic particles), and the like.

  In certain embodiments, at least one characteristic important for the binding affinity of the mutant TNFSF protein is altered when compared to the same characteristic of native TNFSF. In particular, a mutant TNFSF protein with altered receptor affinity is preferred. Also preferred are mutant TNFSF proteins with altered oligomerization affinity for native TNFSF.

  Thus, the present invention is such that mutant TNFSF monomeric proteins preferentially oligomerize to wild type (eg, endogenous) TNFSF monomers but do not substantially agonize the TNFSF receptor. Provided are mutant TNFSF proteins (eg, mixed oligomers containing mutant monomers) with altered binding affinity. In this case, “preferentially” means that at least 10%, more preferably at least 25% of the trimer produced when the equivalent amounts of mutant TNFSF monomer and wild type TNFSF monomer are given mutation It means a mixed trimer of type and wild type TNFSF. At least about 50% is preferred, and at least about 80-90% is particularly preferred. In other words, it is preferable that the mutant TNFSF protein of the present invention has a greater affinity for the wild-type TNFSF protein than the wild-type TNFSF protein. “Substantially does not interact with the TNF receptor” means that the mutant TNFSF protein binds to and activates the TNFSF receptor and / or initiates the TNFSF signaling pathway. It means you can't. In preferred embodiments, a decrease in receptor activation of at least 10% is seen. Preferably greater than 20%, 50%, 76%, 80-90%. “Turn the TNFSF receptor” means that the mutant TNFSF protein enhances receptor signaling. In general, mutant TNFSF proteins that function as agonists of wild-type TNFSF are not preferred.

  Mutant TNFSF proteins can be experimentally tested and confirmed using in vivo and in vitro assays. Suitable assays include, but are not limited to, activity assays and binding assays. Screens that can be used to identify TNFSF variants that are antagonists of TNFSF protein include, but are not limited to, caspase activation, NF-kB nuclear translocation (Wei et al., Endocrinology 142, 1290-1295, (2001)). Or c-Jun (Srivastava et al., JBC 276, 8836-8840 (2001)), including transcription factor activation assay, B cell proliferation assay and IgE secretion assay.

In a preferred embodiment, the binding affinity of the following interactions is measured and compared: 1) mutant TNFSF oligomer formation, 2) wild type TNFSF oligomer formation, 3) binding of mutant TNFSF to the cognate receptor, 4 ) Binding of wild type TNFSF to cognate receptors, 5) binding of mutant TNFSF to attracting receptors, and 6) binding of wild type TNFSF to attracting receptors. Suitable assays include, but are not limited to, a quantitative comparison of kinetic and equilibrium binding constants. Kinetic association rates (K _on ) and dissociation rates (K _off ) and equilibrium binding constants (Kd) can be measured using surface plasmon resonance with a BIAcore instrument according to standard literature procedures [Pearce et al. al., Biochemistry 38: 81-89 (1999)]. Several alternative methods can be used to measure binding affinity and kinetics, including but not limited to proximity assays such as AlphaScreen ^™ (Packard BioScience®).

  The TNFSF variants can also be tested to determine whether mixed oligomers can be formed, including but not limited to mixed trimers. In a preferred embodiment, this is accomplished by labeling native TNFSF and mutant TNFSF with a distinguishable tag, combining native TNFSF and mutant TNFSF, and screening for oligomers containing both types of tags. For example, FLAG-tagged natural TNFSF and His-tagged mutant TNFSF can be combined and a sandwich ELISA can be performed to identify trimers containing both FLAG and His tags. Another alternative is to run the native gel to separate the mixture into separate species and detect using Coomassie staining or Western blot using both anti-FLAG and anti-His tag antibodies. This method relies on the fact that FLAG and His tags significantly disturb protein migration in native gels. Many alternative protocols can also be used to measure mixed trimer formation, as will be apparent to those skilled in the art.

  As outlined above, the present invention provides mutant TNFSF nucleic acids that encode mutant TNFSF polypeptides. A mutant TNFSF polypeptide preferably changes when at least one property is contrasted to the same property of a naturally occurring TNFSF polypeptide. The properties of the mutant TNFSF polypeptide are a result of the present invention.

A “changed property” or grammatical equivalents thereof in the context of a polypeptide, as used herein, is a characteristic of a polypeptide that can be selected or detected and compared to the corresponding property of a naturally-occurring protein. Or it means an attribute. These properties include, but are not limited to: cytotoxic activity, oxidative stability, substrate specificity, substrate binding or catalytic activity, thermal stability, alkaline stability, pH activity profile, resistance to proteolysis Gender, kinetic association (K _on ) and dissociation (K _off ) rates, protein folding, induction of immune response, ligand binding ability, receptor binding ability, secreted ability, ability to be displayed on the cell surface, oligomer Ability to signal, signal transduction, ability to stimulate cell proliferation, ability to inhibit cell growth, ability to induce apoptosis, ability to be modified by phosphorylation or glucosylation, and ability to treat disease.

Unless otherwise specified, the substantial change in any of the listed properties is preferably at least 20%, more preferably 50%, when the properties of the mutant TNFSF polypeptide are compared to those of the naturally produced TNFSF protein. More preferably, it is increased or decreased by a factor of two.
The change in cytotoxic activity is evidenced by a decrease of at least 75% or more in cell death initiated by the mutant TNFSF protein compared to the wild type protein.
The change in binding affinity is evidenced by an increase or decrease in binding affinity for wild type TNF receptor protein or wild type TNFSF by at least 5% or more.

  In a preferred embodiment, the antigenic profile of the mutant TNFSF protein in the host animal is similar to and preferably equivalent to the antigenic profile of the host TNFSF; that is, the mutant TNFSF protein is host to the immune response. Does not significantly stimulate an organism (eg, patient); that is, no immune response is clinically significant, nor is there an allergic reaction or protein neutralization by antibodies. That is, in a preferred embodiment, the mutant TNFSF protein does not contain additional or different epitopes than wild type or naturally occurring TNFSF. As used herein, “epitope” or “determinant” refers to the portion of a protein that produces and / or binds to an antibody. Thus, in most instances, no significant amount of antibody is produced in the native host against the mutant TNFSF protein. In general, this is by not significantly altering surface residues as outlined below, or by not adding amino acid residues on the surface that can be glucosylated (since new glucosylation can result in an immune response). ) Or by not introducing new MHC binding epitopes.

  The mutant protein of the present invention may be shorter or longer than the amino acid sequence shown in FIG. As used herein, “wild type TNFSF” is a natural mammalian protein (preferably human). TNFSF can be polymorphic. Accordingly, in a preferred embodiment, what is included in the definition of a mutant TNFSF protein is a portion or fragment of the sequence set forth herein. A fragment of a mutant TNFSF protein a) shares at least one antigenic epitope; b) has at least the indicated homology; c) and preferably a biological activity of the mutant TNFSF as defined herein. Is considered a mutant TNFSF protein.

  In a preferred embodiment, as outlined below in more detail, in contrast to wild type TNFSF, the mutant TNFSF protein contains even more amino acid changes than those outlined here. Examples include amino acid substitutions introduced to allow soluble expression in E. coli, amino acid substitutions introduced to optimize solution behavior, and introduced to regulate immunogenicity Amino acid substitutions made, but are not limited to these. And, as outlined here, any of the changes depicted here can be combined in any way to form a new mutant TNFSF protein.

  Further, for example, as outlined herein, the addition of epitopes or purification tags, the addition of other fusion sequences, etc. can create mutant TNFSF proteins that are longer than those depicted in the figure. For example, the mutant TNFSF protein of the invention can be fused to other therapeutic proteins or to other proteins such as Fc or serum albumin for pharmacokinetic purposes. See, for example, US Pat. Nos. 5,766,883 and 5,876,969, both of which are hereby incorporated by reference.

  A mutant TNFSF protein can also be identified as encoded by a mutant TNFSF nucleic acid. In the case of nucleic acids, the overall homology of the nucleic acid sequence corresponds to the homology of the amino acids, but takes into account the degeneracy of the genetic code and the codon bias of various organisms. Thus, nucleic acid sequence homology may be lower or higher than that of protein sequences, and low homology is preferred.

  In a preferred embodiment, the mutant TNFSF nucleic acid encodes a mutant TNFSF protein. As will be apparent to those skilled in the art, because of the degeneracy of the genetic code, a very large number of nucleic acids can be generated that all encode the mutant TNFSF protein of the present invention. Thus, once a particular amino acid sequence has been identified, one of skill in the art can create any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the mutant TNFSF.

  The mutant TNFSF proteins and nucleic acids of the invention are preferably recombinant (unless made synthetically). “Nucleic acid” as used herein refers to a molecule comprising either DNA or RNA, or both deoxyribonucleotides and ribonucleotides. This nucleic acid includes genomic DNA, cDNA, mRNA and oligonucleotide, and includes sense and antisense nucleic acids. Such nucleic acids can also contain modifications in the ribose-phosphate backbone to increase the stability and half-life of such molecules in a physiological environment.

  Nucleic acids can be double stranded, single stranded, or contain portions of both double stranded and single stranded sequence. As will be apparent to those skilled in the art, the depiction of a single strand (“Watson”) defines the sequence of the other strand (“Crick”); thus, the sequence shown in FIG. Also includes the complementary strand.

  The definition of the mutant TNFSF protein of the present invention also includes amino acid sequence variants of the mutant TNFSF sequences outlined herein and shown in the figures. That is, the mutant TNFSF protein may contain additional variable positions in addition to those used to generate dominant negative proteins, in contrast to human TNFSF. With respect to “dominant negative variable positions”, these variants belong to one or more of the three groups of substitution, insertion or deletion variants. All variants are usually produced as follows. That is, using cassette or PCR mutagenesis or other techniques well known in the art, nucleotide-directed mutagenesis of the DNA encoding the mutant TNFSF protein to produce DNA encoding the mutant, The DNA is expressed in recombinant cell culture as outlined above. However, mutant TNFSF protein fragments of up to about 100-150 residues can be produced by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by a pre-determined nature of the mutation, ie, a feature that separates the variant from naturally occurring allelic or interspecies changes in the amino acid sequence of the mutated TNFSF protein. This variant typically exhibits qualitatively the same biological activity as the naturally occurring analog, but variants with altered characteristics can also be selected.

  The site or region for introducing an amino acid sequence variation is determined in advance, but the mutation per se need not be determined in advance. For example, to optimize the efficiency of mutation at a given site, random mutagenesis may be performed at the target codon or region and the expressed mutant TNFSF protein screened for the optimal combination of desired activities. . Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Searches for variants are performed for optimal characteristics using each assay for activity of the mutant TNFSF protein, or additional properties (eg, stability) as outlined herein.

  Amino acid substitutions are typically of a single residue; insertions are usually on the order of about 1 to about 20 amino acids, although fairly long insertions can be tolerated. Deletions range from about 1 to about 20 residues, but in some cases the deletion can be even larger.

  Substitutions, deletions, insertions or any combination thereof may be used to arrive at the final derivative. In general, such changes are made to a small number of amino acids to reduce molecular changes (eg, conservative modifications). However, even larger changes can be tolerated in certain circumstances (eg, non-conservative modifications).

  Variants typically exhibit the same biological activity qualitatively as the original mutant TNFSF protein and elicit the same immune response, but if desired, the mutant is characterized by the mutant TNFSF protein Is also selected to modify. Alternatively, the mutant can be designed such that the biological activity of the mutant TNFSF protein is altered. For example, glycosylation sites can be altered or removed. Similarly, biological function may be altered; for example, in certain instances it may be desirable to have stronger or weaker TNFSF activity.

  Mutant TNFSF proteins and nucleic acids of the invention can be made in a number of ways. Individual nucleic acids and proteins can be made by methods known in the art and outlined below. Alternatively, a library of mutant TNFSF proteins can be created for testing.

  In a preferred embodiment, a set or library of mutant TNFSF proteins can be generated in a number of ways known to those skilled in the art.

  In a preferred embodiment, different protein members of a mutant TNFSF library can be chemically synthesized. This is particularly useful when the designed protein is short, preferably less than 150 amino acids long, more preferably less than 100 amino acids, and particularly preferably less than 50 amino acids, but as known in the art, Longer proteins can be made chemically or enzymatically. See, for example, Wilken et al., Curr. Opin. Biotechnol. 9: 412-26 (1998), which is hereby incorporated by reference.

  In a preferred embodiment, library sequences are used to create nucleic acids, such as DNA encoding member sequences, which are then optionally hosted, particularly for longer proteins or proteins for which larger samples are desired. It can be cloned, expressed and assayed in cells. Thus, nucleic acids, particularly DNA, encoding the protein sequence of each member can be made. This is done using known methods. The selection of codons, appropriate expression vectors, and appropriate host cells will depend on a number of factors and can be easily optimized as needed.

  In a preferred embodiment, multiple PCR reactions using pooled oligonucleotides are performed when creating a variant library, eg, to test for antagonist activity and / or other desired activity as outlined herein. . In this embodiment, overlapping oligonucleotides corresponding to the full-length gene are synthesized. These oligonucleotides may also represent all of the different amino acids or subsets at each variable position. In a preferred embodiment, these oligonucleotides are pooled in equal proportions and multiple PCR reactions are performed to create a full-length sequence containing the combination of mutations defined by the library. In addition, this can be done using error-prone PCR methods.

  In a preferred embodiment, different oligonucleotides are added in relative amounts corresponding to the probability distribution table, as described in USSN 10 / 218,102. In this way, multiple PCR reactions result in a full-length sequence having the desired combination of mutations in the desired proportion.

  In a preferred embodiment, each overlapping oligonucleotide contains only one position to be changed; in another embodiment, mutation positions are too close to each other to make this possible, Mutations are used to complete all possible recombination. That is, each oligo can contain a single position that is mutated, or one or more codons that are mutated. The multiple positions that are mutated must be close together in the sequence to prevent the oligo length from becoming impractical. By including or excluding an oligonucleotide encoding a particular combination of mutations for multiple mutation positions on an oligonucleotide, the combination can be included or excluded in the library. For example, as discussed herein, there may be a correlation between variable positions; that is, if position X is a residue, position Y must be a specific residue (or Must not). These sets of variable positions are sometimes referred to herein as “clusters”. When a cluster contains residues in close proximity to each other and can therefore be present on a single nucleotide primer, the cluster can be set to a “good” correlation to eliminate bad combinations that can reduce the effectiveness of the library. However, if the cluster residues are separated in the sequence and thus will be present on different oligonucleotides for synthesis, the residues are set to “good” correlation or variable residues. It is desirable to eliminate it completely as a group. In another embodiment, the library can be created in several stages so that cluster mutations appear exclusively together. Experimental libraries by identifying mutant clusters and placing them on the same oligonucleotide or removing them from the library, or generating a library in several steps while preserving the cluster Can be quite rich in properly folded proteins. Cluster identification can be done, for example, using known pattern recognition methods, comparing mutation frequencies, or analyzing the energy of experimentally generated sequences (eg, if the interaction energy is high, the position is correlated) It can be implemented in a number of ways such as use. These correlations are both positional correlations (eg, variable positions 1 and 2 always change together or never change together), even if they are sequence correlations (eg, if there is residue A at position 1, position 2 always There may be a residue B). Pattern discovery in Biomolecular Data: Tools, Techniques, and Applications; edited by Jason TL Wang, Bruce A. Shapiro, Dennis Shasha.New York: Oxford University, 1999; Andrews, Harry C. Introduction to mathematical techniques in pattern recognition; New York , Wiley-lnterscience [1972]; Applications of Pattern Recognition; Editor, KS Fu. Boca Raton, Fla. CRC Press, 1982; Genetic Algorithms for Pattern Recognition; edited by Sankar K. Pal, Paul P. Wang. Boca Raton: CRC Press, c1996; Pandya, Abhijit S., Pattern recognition with neural networks in C ++ / Abhijit S. Pandya, Robert B. Macy. Boca Raton, Fla .: CRC Press, 1996; Handbook of pattern recognition & computer vision / edited by CH Chen, LF Pau, PSP Wang. 2nd ed. Singapore; River Edge, NJ: World Scientific, c1999; Friedman, Introduction to Pattern Recognition: Statistical, Structural, Neural, and Fuzzy Logic Approaches; River Edge, NJ: World Scientific, c1999 , Series title: Series in machine perception and artificial i ntelligence; vol. 32; (all incorporated herein by reference). In addition, programs used for consensus motif searches can be used as well.

  Oligonucleotides with codon insertions or deletions can be used to create libraries that express proteins of different lengths. In particular, computerized sequence screening for insertions or deletions can result in a secondary library defining proteins of different lengths. They can be expressed by a library of oligonucleotides of various lengths pooled.

  In another preferred embodiment, the mutant TNFSF protein of the invention is created by shuffling this family (eg, a set of variants); ie, a higher sequence (when using a ranked list) Some sets can be shuffled with or without error-prone PCR. “Shuffling” in this context means recombination of related sequences, generally in a random manner. This is defined and illustrated in US Pat. Nos. 5,830,721, 5,811,238, 5,605,793, 5,837,458 and PCT / US / 19256. All of which are hereby incorporated by reference in their entirety. This set of sequences may be derived from an artificial set; for example, a probability table (eg, created using SCMF) or a set of Monte Carlo. Similarly, the “family” may be the upper 10 and lower 10 sequences, the upper 100 sequences, and the like. This can also be done using error-prone PCR.

  Accordingly, in a preferred embodiment, in silico shuffling is performed using the computer processing methods described herein. That is, starting with two libraries or two sequences, random recombination of sequences can be generated and evaluated.

  In a preferred embodiment, the mutant TNFSF protein is a chimera formed from two or more naturally occurring TNFSF proteins. In a particularly preferred embodiment, the chimera is formed by linking a receptor contact region from one or more naturally occurring TNFSF proteins to the amino acid sequence of another naturally occurring TNFSF protein.

  In a preferred embodiment, error-prone PCR is performed to generate a library of mutant TNFSF proteins. See U.S. Pat. Nos. 5,605,793, 5,811,238, and 5,830,721, all of which are incorporated herein by reference. This can be done for the optimal sequence or top member of the library, or some other artificial set or family. In this embodiment, the gene of the optimal sequence found by computer screening of the primary library can be synthesized. Then, error-prone PCR is performed with the optimally sequenced gene in the presence of an oligonucleotide encoding a mutation at the mutation position of the library (biased oligonucleotide). The addition of these oligonucleotides will yield a bias that favors incorporating the mutation into the library. Alternatively, only the oligonucleotides for certain mutations can be used to bias the library.

  In a preferred embodiment, gene shuffling by error-prone PCR is performed on optimally sequenced genes in the presence of biased oligonucleotides to generate a DNA sequence library that reflects the percentage of mutations found in the mutant TNFSF library. Can be created. Selection of biased oligonucleotides can be done in a variety of ways; one can select based on that frequency bias, ie, one can use oligonucleotides that encode high mutation frequency positions; or to increase diversity, Oligonucleotides containing the most susceptible positions can be used; when ranking secondary libraries, several top score positions can be used to generate biased oligonucleotides; You can select one with several high scores, one with several low scores, and so on. What is important is to generate new sequences based on preferred variable positions and sequences.

  In a preferred embodiment, PCR using wild type genes or other genes may be used, as schematically shown in the figure. In this embodiment, a starting gene is used; although not required, in general, this gene is usually a wild type gene. In some cases, this can be a gene encoding a global optimal sequence, or other sequence in the list, or a consensus sequence obtained, for example, by aligning homologous sequences from different organisms. In this embodiment, oligonucleotides are used that correspond to the mutated positions and contain different amino acids in the library. As is known in the art, PCR is performed using PCR primers at the ends. This has two advantages. First, this generally results in fewer oligonucleotides and fewer errors. Second, when using a wild type gene, there is an experimental advantage in that it does not need to be synthesized.

  Various expression vectors are made using the nucleic acids of the invention encoding mutant TNFSF proteins. The expression vector can be a self-replicating extrachromosomal vector or a vector that integrates into the host genome. In general, these expression vectors include transcriptional and translational regulatory nucleic acids operably linked to the nucleic acid encoding the mutant TNFSF protein. The term “control sequence” refers to a DNA sequence that is required for expression in a particular host organism of an operably linked coding sequence. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

  Nucleic acids are “operably linked” when placed into a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA is operably linked to the DNA of the polypeptide if it is expressed as a preprotein that participates in secretion of the polypeptide; a promoter or enhancer is encoded by If it affects the transcription of the sequence, it is operably linked to that sequence; or the ribosome binding site can function to the coding sequence if it is positioned to facilitate translation. They are linked together.

  In a preferred embodiment, it is desirable to replace this naturally occurring secretory leader sequence if the endogenous secretory sequence leads to low level secretion of the naturally occurring protein or mutant TNFSF protein. In this embodiment, an irrelevant secretory leader sequence is operably linked to the mutant TNFSF-encoding nucleic acid, leading to increased protein secretion. Thus, any secretory leader sequence that results in enhanced secretion of the mutant TNFSF protein when compared to secretion of TNFSF and its secretory sequence is desirable. Suitable secretory leader sequences that direct protein secretion are known in the art.

  In another preferred embodiment, the naturally occurring protein or secretory leader sequence of the protein is removed by techniques known in the art and then expressed, resulting in intracellular accumulation of the recombinant protein.

  In general, “operably linked” means that the linked DNA sequences are contiguous and, in the case of a secretion leader, are contiguous and in reading frame. However, enhancers do not have to be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. Transcriptional and translational regulatory nucleic acids are generally suitable for host cells used to express the fusion protein; for example, transcriptional and translational regulatory nucleic acid sequences derived from Bacillus are preferably used for expression of the fusion protein in Bacillus. . Numerous types of appropriate expression vectors, and suitable control sequences are known in the art for a variety of host cells.

  In general, transcriptional and translational control sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Absent. In a preferred embodiment, the control sequences include a promoter and transcription start and stop sequences.

  A promoter sequence encodes either a constitutive or inducible promoter. The promoter can be either a naturally occurring promoter or a hybrid promoter. Hybrid promoters that combine elements of more than one promoter are also known in the art and are useful in the present invention. In a preferred embodiment, the promoter is a strong promoter that allows for high expression in cells, particularly mammalian cells, such as a CMV promoter, especially in combination with Tet regulatory elements.

  In addition, the expression vector may contain additional elements. For example, an expression vector has two replication systems and can therefore be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. To. Furthermore, to incorporate an expression vector, the expression vector contains at least one sequence that is homologous to the host cell genome, and preferably two homologous sequences that flank the expression construct. This integrating vector can be directed to a specific locus in the host cell by selecting appropriate homologous sequences for inclusion in the vector. Integration vector constructs are well known in the art.

  In addition, in a preferred embodiment, the expression vector contains a selectable marker gene that allows for selection of transfected host cells. Selection genes are well known in the art and will vary with the host cell used.

  Preferred expression vector systems are the retroviral vector systems generally described in PCT / US97 / 01019 and PCT / US97 / 01048, both of which are hereby incorporated by reference.

  In a preferred embodiment, the expression vector comprises the above-described components and a gene encoding a mutant TNFSF protein. Those skilled in the art will appreciate that all combinations are possible and therefore, as used herein, consist of one or more vectors that may or may not be retroviruses. The combination of components is referred to herein as a “vector composition”.

Mutant TNFSF nucleic acids are introduced into cells alone or in combination with expression vectors. As used herein, “introducing” or grammatical equivalent means that the nucleic acid enters the cell in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely defined by the target cell type as discussed below. Examples of methods include CaPO ₄ precipitation, liposome fusion, Lipofectin®, electroporation, viral infection and the like. The mutant TNFSF nucleic acid can be stably integrated into the genome of the host cell (eg, by retroviral introduction as outlined below) or can be transiently or stably present in the cytoplasm (ie, through the use of conventional plasmids). , By using standard control sequences, selectable markers, etc.).

  The mutant TNFSF protein of the present invention is cultured in a host cell transfected with an expression vector containing a nucleic acid encoding the mutant TNFSF protein under conditions suitable for inducing or inducing the expression of the mutant TNFSF protein. It is produced by. Appropriate conditions for expression of the mutant TNFSF protein will vary with the choice of the expression vector and the host cell and will be readily ascertainable by one skilled in the art through routine experimentation. For example, constitutive promoters in expression vectors require optimization of host cell growth and proliferation, and the use of inducible promoters requires growth conditions suitable for induction. Furthermore, in some embodiments, the timing of recovery is important. For example, the baculovirus system used in insect cell expression is a lytic virus, so the choice of harvest time can be critical to product yield.

  Suitable host cells include yeast, bacteria, archaea, fungi, and animal cells including insect and mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, Pichia Pastoris and the like.

  In a preferred embodiment, the mutant TNFSF protein is expressed in mammalian cells. Mammalian expression systems are also known in the art and include retroviral systems. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating downstream (3 ′) transcription of the coding sequence of the fusion protein into mRNA. A promoter has a transcription initiation region which is usually located proximal to the 5 ′ end of the coding sequence and a TATA box which uses 25-30 base pairs located upstream of this transcription initiation location. This TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct position. A mammalian promoter will further comprise an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. The upstream promoter element determines the rate of transcription initiation and can act in either direction. Particularly useful as mammalian promoters are promoters derived from mammalian viral genes. This is because viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and CMV promoter.

  Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3 'to the translation stop codon and are thus adjacent to the coding sequence along with the promoter element. The 3 ′ end of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminators and polyadenylation signals include those derived from SV40.

  Methods for introducing exogenous nucleic acid into mammalian hosts and other hosts are well known in the art and will vary with the host cell used. Methods include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. . As outlined herein, a particularly preferred method utilizes the retroviral infection outlined in PCTUS 97/01019 (incorporated herein by reference).

  As will be appreciated by those skilled in the art, the types of mammalian cells used in the present invention can vary widely. Basically, any mammalian cell can be used, mouse, rat, primate and human cells being particularly preferred, but as will be appreciated by those skilled in the art, all by modification of the system by pseudotyping Eukaryotic cells, preferably higher eukaryotic organisms. As described in more detail below, the screen is set up such that the cells exhibit a selectable phenotype in the presence of the bioactive peptide. As detailed below, cell types associated with a wide variety of disease symptoms are particularly useful as long as appropriate screening can be set up to select cells that exhibit altered phenotypes due to the presence of the peptide in the cell.

  Thus, suitable cell types include all types of tumor cells (especially melanoma, myeloid leukemia, lung, breast, ovary, colon, kidney, prostate, pancreas and testicular carcinoma), cardiomyocytes, endothelial cells, epithelium Cells, lymphocytes (T cells and B cells), mast cells, eosinophils, intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, hematopoiesis, nerve, skin, lung, kidney, liver and myocyte stem cells These include stem cells (for use in screening differentiation and dedifferentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes It is not necessarily limited to. Suitable cells further include known research cells, including but not limited to Jurkat T cells, NIH3T3 cells, CHO, Cos and the like. See ATCC cell line catalog (incorporated herein by reference).

In certain embodiments, the cells can be further genetically engineered. That is, it includes exogenous nucleic acids other than mutant TNFSF nucleic acids.
In a preferred embodiment, the mutant TNFSF protein is expressed in a bacterial system. Bacterial expression systems are well known in the art.

  A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating downstream (3 ') transcription of the mutant TNFSF protein coding sequence into mRNA. A bacterial promoter has a transcription initiation region which is usually located proximal to the 5 ′ end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Bacteriophage derived promoters can also be used and are known in the art. In addition, synthetic and hybrid promoters are also useful; for example, the tac promoter is a hybrid of trp and lac promoter sequences. In addition, bacterial promoters can include naturally produced promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.

  In addition to a functional promoter sequence, an effective ribosome binding site is desirable. In E. coli, this ribosome binding site is called the Shine-Delgarno (SD) sequence and contains a start codon and a 3-9 nucleotide long sequence 3-11 nucleotides upstream of the start codon. The expression vector also includes a signal peptide sequence that provides for secretion of the mutant TNFSF protein in bacteria. The signal sequence typically encodes a signal peptide consisting of hydrophobic amino acids that direct secretion of the protein from the cell, as is well known in the art. Proteins are secreted into the growth medium (Gram positive bacteria) or into the periplasmic space located between the outer and inner membranes of cells (Gram negative bacteria). For bacterial expression, a bacterial secretory leader sequence operably linked to the mutant TNFSF-encoding nucleic acid is usually preferred.

  Bacterial expression vectors can also include a selectable marker gene that allows selection of the transfected bacterial strain. Suitable selection genes include genes that make bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.

  These components are assembled into an expression vector. Expression vectors for bacteria are well known in the art and include, among others, vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, Streptococcus lividans.

  Bacterial expression vectors are transfected into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation and the like.

  In certain embodiments, the mutant TNFSF protein is produced in insect cells. Expression vectors for transfection of insect cells, particularly expression vectors based on baculovirus, are well known in the art.

  In a preferred embodiment, the mutant TNFSF protein is produced in yeast cells. Yeast expression systems are well known in the art, including Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytic Is done. Preferred promoter sequences for expression in yeast include inducible GAL1,10 promoter, alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, phosphofructokinase , Promoters derived from 3-phosphoglycerate tomase, pyruvate kinase, and acid phosphatase genes. Yeast selectable markers include ADE2, HIS4, LEU2, TRP1, ALG7 conferring tunicamycin resistance, the neomycin phosphotransferase gene conferring G418 resistance, and the CUP1 gene that allows yeast to grow in the presence of copper ions.

  In a preferred embodiment, the modified TNFSF variant is covalently coupled to at least one additional TNFSF variant via a linker to improve the dominant negative effect of the modified domain. A number of strategies can be used to covalently link modified receptor domains together. These include linkers such as polypeptide linkages between the N- and C-termini of two domains, linkages between disulfide bonds between monomers, linkages via chemical cross-linking reagents, It is not limited. Alternatively, the N-terminus and C-terminus can be covalently linked by deleting part of the N-terminus and / or C-terminus and linking the remaining fragments via a linker, or by directly linking the fragments .

  A “linker”, “linker sequence”, “spacer”, “tethered sequence” or grammatical equivalents thereof, as used herein, connects two molecules and often places the two molecules in a preferred configuration. Means a molecule or group of molecules (such as monomers or polymers) that are useful for In certain aspects of this embodiment, the linker is a peptide bond. The choice of a suitable linker for a particular case connecting two polypeptide chains depends on various parameters. For example, the nature of two polypeptide chains (eg, whether they naturally multimerize (eg, whether they form a dimer)), if known from the 3D structure determination, Such as the distance between the connected N-terminus and C-terminus, and / or the stability of the linker against proteolysis and oxidation. In addition, the linker may contain amino acid residues that provide flexibility. Thus, the linker peptide predominantly contains the following amino acid residues: Gly, Ser, Ala or Thr. These linked TNFSF proteins have unnatural hydrodynamic properties (ie they form constitutive dimers) and thus interact efficiently with other naturally occurring TNFSF proteins. , Forming a dominant negative heterotrimer.

  The linker peptide has the appropriate length to link the two TNFSF mutant monomers in such a way that they are correctly positioned relative to each other and retain the desired activity as an antagonist of the native TNFSF protein. Should have. Suitable lengths for this purpose include at least 1 and no more than 30 amino acid residues. Preferably, the linker is about 1 to 30 amino acids long and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 amino acid long linkers are preferred. See also WO01 / 25277, which is hereby incorporated by reference in its entirety.

  Furthermore, the amino acid residues selected for inclusion in the linker peptide should exhibit properties that do not significantly interfere with the activity of the polypeptide. Thus, the linker peptide as a whole should not exhibit a charge contrary to the activity of the polypeptide, or should interfere with internal folding, or accept one or more amino acid residues of the monomer and accept No bonds or other interactions should be formed that significantly interfere with the binding of body monomer domains.

  As will be appreciated by those skilled in the art, useful linkers include glycine-serine polymers (eg, (GS) n, (GSGGS) n, (GGGGGS) n, and (GGGS) n), where n is an integer of at least 1. Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as shaker-type potassium channel tethers, and a number of various other flexible linkers. A glycine-serine polymer is preferred. Because both of these amino acids are relatively unstructured and can therefore serve as a neutral bridge between the components. Secondly, serine is hydrophilic and thus can solubilize what may be a globular glycine chain. Third, similar chains have been shown to be effective in linking subunits of recombinant proteins such as single chain antibodies.

Suitable linkers can also be identified by searching known three-dimensional structural databases for naturally occurring motifs that can bridge the gap between two polypeptide chains. Another way to obtain a suitable linker is by optimizing a simple linker (eg (Gly4Ser) n) through random mutagenesis. Alternatively, once a suitable polypeptide linker has been defined, amino acids that interact more optimally with the domains to be linked can be selected by application of PDF ^™ technology to create additional linker polypeptides. Other types of linkers that can be used in the present invention include artificial polypeptide linkers and inteins. In another preferred embodiment, a disulfide bond is designed to link two receptor monomers at the intermonomer contact site. In one aspect of this embodiment, the two receptors are linked at a distance of <5 angstroms. In addition, the mutant TNFSF polypeptides of the invention can be further fused to other proteins, if desired, for example to increase expression or stabilize the protein.

  In one embodiment, the mutant TNFSF nucleic acids, proteins and antibodies of the invention are labeled with a label outside of the backbone. As used herein, “labeling” means that a compound has at least one element, isotope or chemical compound attached to allow detection of the compound. In general, labels are divided into three classes: a) isotope labels, which can be radioactive or heavy isotopes; b) immunolabels, which can be antibodies or antigens; and c) colored or fluorescent dyes. The label may be incorporated into the compound at any position.

  Once generated, the mutant TNFSF protein can be modified. Covalent and non-covalent modifications of the protein are within the scope of the present invention. Such modifications can be introduced into the mutant TNFSF polypeptide by reacting the target amino acid residue of the polypeptide with an organic derivatization reagent capable of reacting with a selected side chain or terminal residue.

  One type of covalent modification involves reacting a target amino acid residue of a mutant TNFSF polypeptide with an organic derivatization reagent capable of reacting with a selected side chain or N or C-terminal residue of the mutant TNFSF polypeptide. Including that. For example, as detailed below, to crosslink a mutant TNFSF protein to a water-insoluble support matrix or surface for use in a purification method or screening assay for anti-mutant TNFSF antibodies, Derivatization with sexual reagents is useful. Commonly used cross-linking reagents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, disuccin Homobifunctional imidoesters including imidyl esters such as 3,3′-dithiobis (succinimidylpropionate), difunctional maleimides such as bis-N-maleimide-1,8-octane and Reagents such as methyl-3-[(p-azidophenyl) dithio] propioimidate are included.

  Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, lysine, arginine, and Methylation of the amino group of the histidine side chain [TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of N-terminal amine, and optional C Includes amidation of the terminal carboxyl group.

  Other types of covalent modifications of mutant TNFSF polypeptides encompassed within the scope of the present invention include alterations in the natural glycosylation pattern of the polypeptide. “Modification of native glycosylation pattern” for the purposes of the present invention removes one or more carbohydrate moieties found in a native sequence variant TNFSF polypeptide and / or is present in a native sequence variant TNFSF polypeptide. It is intended to mean adding one or more glycosylation sites.

  Addition of glycosylation sites to the mutant TNFSF polypeptide can be accomplished by changing its amino acid sequence. This alteration may be achieved, for example, by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) to the native sequence or mutant TNFSF polypeptide. The mutant TNFSF amino acid sequence can optionally be altered through changes at the DNA level. In particular, by mutating the DNA encoding the mutant TNFSF polypeptide with a preselected base so as to generate a codon that will be translated into the desired amino acid.

  Addition of an N-linked glycosylation site to a mutant TNFSF polypeptide can be accomplished by changing its amino acid sequence. Changes can be made, for example, by adding or substituting one or more asparagine residues to the native sequence or mutant TNFSF polypeptide. Modifications include, for example, N—X—Y, where X is any amino acid other than proline, and Y is preferably threonine, serine or cysteine, canonical linkage This can be done by introducing a type glycosylation site. Another means of increasing the number of carbohydrate moieties in the mutant TNFSF polypeptide is by chemically or enzymatically coupling glycosides to the polypeptide. Such methods are described in the art, for example in WO 87/05330 published September 11, 1987 and Aplin and Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981).

  Removal of the carbohydrate moiety present in the mutant TNFSF polypeptide can be accomplished chemically, enzymatically, or by mutagenic substitution of a codon encoding the amino acid residue that is the target of glycosylation. Chemical deglycosylation techniques are known in the art, for example, Hakimuddin, et al., Arch. Biochem. Biophys., 259: 52 (1987) and Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of the carbohydrate portion of the polypeptide can be achieved by the use of various endo- and exo-glycosidases, as described in Thotakura et al., Meth. Enzymol., 138: 350 (1987).

  Such induced moieties may improve solubility, absorbability, and blood brain barrier permeability, biological half-life, and the like. Such a portion or modification of the mutant TNFSF polypeptide may alternatively eliminate or reduce such unwanted side effects of the protein. Portions that can mediate such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

  Other types of covalent modifications of mutant TNFSF include mutant TNFSF polypeptides, such as US Pat. Nos. 4,640,835; 4,496,689; 4,301,144; No. 670,417; No. 4,791,192; No. 4,179,337; No. 5,183,550, polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylene, etc. Linking to one of a variety of non-proteinaceous polymers. These non-proteinaceous polymers can also be used to increase the ability of mutant TNFSF to disrupt receptor binding and / or in vivo stability.

  In other preferred embodiments, cysteines are designed in mutant or wild type TNFSF to (a) introduce a labeling site for characterization and (b) introduce a PEGylation site. For example, labels that can be used are well known in the art and include, but are not limited to, biotin, tags and fluorescent labels (eg, fluorescein). These labels can also be used to achieve characterization in various assays, as is well known in the art. As is well known in the art, various coupling chemistries can be used to achieve PEGylation. Examples include, but are not limited to, Shearwater and Enzon techniques that allow modification with primary amines, including but not limited to lysine groups and N-termini. Kinstler et al, Advanced Drug Deliveries Reviews, 54, 477-485 (2002) and MJ Roberts et al, Advanced Drug Delivery Reviews, 54, 459-476 (2002), both of which are hereby incorporated by reference. Please refer to.

  Other modifications may be made to the mutant TNFSF protein of the invention. This includes protein modifications that enhance stability, dose management (eg, amphiphilic polymers (see WO0141812A2, sold by Nobex Corporation), clearance (eg, amphiphilic moieties that affect binding to PEG, HSA), etc. Is included.

  The optimal site for modification can be selected using a variety of criteria including visual inspection, structural analysis, sequence analysis and molecular simulation. Individual residues can be analyzed to identify mutation sites that do not disrupt the monomer structure. The distance from each side chain of the monomer to the other subunit is then calculated to ensure that chemical modification does not disturb the oligomerization. Receptor binding disruption may occur and may be advantageous for the activity of the TNFSF variants of the invention.

  In another preferred embodiment, either the N- or C-terminal part of the wild-type TNFSF monomer is deleted while still allowing the TNFSF molecule to fold correctly. In addition, these modified TNFSF proteins substantially lack receptor binding and / or activation and optionally interact with other wild-type TNFSF molecules or modified TNFSF proteins to trimer as described above. (Or other oligomers) may be formed.

  In addition, removal or deletion of about 1 to about 55 amino acids from the N- or C-terminus, or both, is preferred. More preferred embodiments include N-terminal deletions beyond residue 10, with the first 47 N-terminal amino acid deletions being more preferred. The deletion of the C-terminal leucine is an alternative embodiment.

In another preferred embodiment, wild type TNFSF or a variant produced according to the present invention can be rearranged in a circular fashion. All natural proteins have an amino acid sequence starting at the N-terminus and ending at the C-terminus. TNFSF that is cyclized or circularly rearranged, linking the N and C termini and retaining or improving biological properties relative to the wild type protein (eg, increased stability and activity) Can create proteins. In the case of a TNFSF protein, a new set of N and C termini is usually created at amino acid positions inside the primary structure of the protein, and the original N and C termini are passed through a peptide linker consisting of 0 to 30 amino acids in length. Connect (some amino acids located near the native end may be removed to match the linker design). In a preferred embodiment, the new N and C termini are located in irregular secondary structure elements such as loops and turns, such that the stability and activity of the new protein is similar to that of the native protein. The circularly rearranged TNFSF protein can be further PEGylated or glycosylated. In a further preferred embodiment, PDA ^™ technology can be used to further optimize TNFSF variants, particularly in regions created by circular permutation. These include the new N and C termini, as well as the native terminator and linker peptides.

  Various techniques can be used to reorder proteins. US 5,981,200; Maki K, Iwakura M., Seikagaku. 2001 Jan; 73 (1): 42-6; Pan T., Methods Enzymol. 2000; 317: 313-30; Heinemann U, Hahn M., Prog Biophys Mol Biol 1995; 64 (2-3): 121-43; Harris ME, Pace NR, Mol Biol Rep. 1995-96; 22 (2-3): 115-23; Pan T, Uhlenbeck OC., 1993 Mar 30; 125 (2): 111-4; Nardulli AM, Shapiro DJ. 1993 Winter; 3 (4): 247-55, EP 1098257 A2; WO 02/22149; WO 01/51629; WO 99/51632; Hennecke, et al , 1999, J. Mol. Biol., 286, 1197-1215; Goldenberg et al J. Mol. Biol 165, 407-413 (1983); Luger et al, Science, 243, 206-210 (1989); and See Zhang et al., Protein Sci 5, 1290-1300 (1996). All of which are incorporated herein by reference.

In addition, a completely cyclic TNFSF can be produced in which the protein contains no termini. This is achieved using intein technology. In this way, peptides can be cyclized, in particular inteins can be utilized to achieve cyclization.
Circularization and circular permutation can be used to generate the dominant negative activity of the TNFSF protein of the invention.

  The mutant TNFSF polypeptides of the present invention can also be modified in a way that forms a chimeric molecule comprising the mutant TNFSF polypeptide fused to another heterologous polypeptide or amino acid sequence. In certain embodiments, such chimeric molecules comprise a fusion of a mutant TNFSF polypeptide and a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. This epitope tag is generally located at the amino or carboxyl terminus of the mutant TNFSF polypeptide. The presence of such an epitope-tagged form of a mutant TNFSF polypeptide can be detected using an antibody against the tag polypeptide. The provision of an epitope tag also allows the mutant TNFSF polypeptide to be easily purified by affinity purification using an anti-tag antibody or other type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of a mutant TNFSF polypeptide and an immunoglobulin or a specific region of an immunoglobulin. For the bivalent form of the chimeric molecule, such a fusion can be to the Fc or Fab region of an IgG molecule. Other fusion components include human serum albumin (HSA), hydrophobic peptides, fatty acid molecules, labels, and the like.

  Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (polyhis) or poly-histidine-glycine (poly-his-gly) tag; fluHA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8: 2159- 2165 (1988)]; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)]; and herpes simplex virus A glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)] are included. Other tag polypeptides include Flag peptide [Hopp et al., BioTechnology 6: 1204-1210 (1988)]; KT3 epitope peptide [Martin et al., Science 255: 192-194 (1992)]; tubulin epitope Peptide [Skinnere et al., J. Biol. Chem. 266: 15163-15166 (1991)]; and T7 gene 10 protein peptide label [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA 87: 6393 −6397 (1990)].

  In a preferred embodiment, the mutant TNFSF protein is purified or isolated after expression. The mutant TNFSF protein can be isolated or purified in various ways known to those skilled in the art, depending on what other components are present in the sample. Standard purification methods include electrophoresis, molecular, immunological techniques and chromatographic techniques including ion exchange, hydrophobicity, affinity and reverse phase HPLC chromatography and chromatofocusing. For example, the mutant TNFSF protein can be purified using a standard anti-library antibody column. Ultrafiltration and diafiltration techniques coupled with protein concentration are also useful. For general guidance on suitable purification techniques, see Scopes, R .; Protein Purification, Springer-Verlag, NY (1982). The degree of purification required will vary depending on the application of the mutant TNFSF protein. In some instances no purification will be necessary.

  In a preferred embodiment, the mutant TNFSF protein of the invention is produced and purified separately from the naturally produced TNFSF protein to be antagonized. That is, a mutant monomer is prepared and introduced into a wild-type protein in the form of a monomer or oligomer. For example, a preferred method involves administration of a mutant monomer to a patient, whereby the mutant monomer “exchanges” into a homo-oligomer (generally a trimer) to produce a mixed oligomer and receive It leads to a decrease in body activation. In another embodiment, the mutant oligomer can be administered and then replaced. However, in some embodiments, such as gene therapy applications, the mutant TNFSF protein can be produced substantially simultaneously with the naturally produced TNFSF target.

  Once made, the mutant TNFSF proteins and nucleic acids of the present invention have applications in numerous applications. In a preferred embodiment, the mutant TNFSF protein is administered to a patient to treat a TNFSF-related disease.

  As used herein, “TNFSF-related disease” or “TNFSF responsive disorder” or “symptom” means a disorder that can be ameliorated by administration of a pharmaceutical composition comprising a mutant TNFSF protein. Includes but is not limited to autoimmunity, inflammatory and immune disorders. Mutant TNFSF protein is a major effector in the pathology of immunoregulatory diseases.

  In a preferred embodiment, the mutant TNFSF protein is, for example, congestive heart failure (CHF), vasculitis, rosecea, acne, eczema, myocarditis and other symptoms of myocardium, systemic lupus erythematosus, diabetes Spondylosis, synovial fibroblasts, bone marrow stroma; bone loss; Paget's disease, giant cell tumor of the bone; multiple myeloma; breast cancer; disuse osteopenia; Periodontal disease, Gaucher disease, Langerhans cell histiocytosis, spinal cord injury, acute septic arthritis, osteomalacia, Cushing syndrome, monoostotic fibrous dysplasia, polyostotic fibrous dysplasia Formation, periodontal reconstruction, and fracture; sarcoidosis; multiple myeloma; osteolytic osteosarcoma, breast cancer, lung cancer, kidney cancer and rectal cancer; bone metastasis, bone pain management, and body Malignant hypercalcemia, ankylosing spondylitis and other spondyloarthropathy; transplant rejection, viral infections, hematopoiesis and neoplastic symptoms such as Hodgkin lymphoma; non-Hodgkin lymphoma (Burkitt lymphoma, small lymphocytic lymphoma) / Chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-type lymphoma, marginal zone lymphoma, ciliary cell leukemia and lymphocytoplasmic leukemia), lymphocyte progenitor cell Tumors (including B cell acute lymphoblastic leukemia / lymphoma and T cell acute lymphoblastic leukemia / lymphoma), thymoma, mature T and NK cell tumors (peripheral T cell leukemia, adult T cell leukemia / T cell lymphoma and Large granular lymphocytic leukemia), Langerhans cell histiocytosis, bone marrow neoplasia (acute myeloid leukemia (maturation observed) AML, undifferentiated AML, acute promyelocytic leukemia, acute myelomonocytic leukemia and acute monocytic leukemia), myelodysplastic syndrome, and chronic myeloproliferative disorder (including chronic myelogenous leukemia) Tumors of the central nervous system, such as brain tumors (glioma, neuroblastoma, astrocytoma, myeloma, ependymoma and retinoblastoma), solid tumors (nasopharyngeal carcinoma, basal cell tumor, pancreas) Cancer, bile duct cancer, Kaposi's sarcoma, testicular cancer, uterine, vaginal or cervical cancer, ovarian cancer, primary liver cancer or endometrial cancer), and vascular tumors (angiosarcoma and vascular ectocytoma), osteoporosis , Hepatitis, HIV, AIDS, spondyloarthritis, rheumatoid arthritis, inflammatory bowel disease (IBD), sepsis and septic shock, Crohn's disease, psoriasis, scleroderma, graft-versus-host disease (GVHD), islet allograft rejection, Blood evil Treating tumors (such as multiple myeloma (MM), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML)), cancer, and inflammation associated with tumors, peripheral nerve injury or demyelinating diseases Used for.

  As used herein, “therapeutically effective dose” means a dose that exhibits the intended effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. In a preferred embodiment, a dose of about 0.01 to about 50 μg / kg is used and is administered either intravenously or subcutaneously. As known in the art, degradation of mutant TNFSF protein, systemic versus local delivery, and the rate of new protease synthesis, as well as age, weight, general health, sex, diet, administration time, drug interactions and conditions Adjustment for severity may be necessary. These will be confirmed by those skilled in the art through routine experimentation.

  “Patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. This method is therefore applicable to both human therapy and veterinary applications. In a preferred embodiment, the patient is a mammal, and in a most preferred embodiment, the patient is a human.

  In the present invention, the term “treatment” is meant to encompass therapeutic treatment for a disease or disorder, as well as prevention or suppression methods. Thus, for example, successful administration of a mutant TNFSF protein prior to the onset of a disease results in a “treatment” of the disease. As another example, successful administration of a mutant TNFSF protein after onset of clinical symptoms of the disease to counter the symptoms of the disease includes “treatment” of the disease. “Treatment” also includes administering a mutant TNFSF protein after the appearance of the disease to eradicate the disease. Successful administration after onset and after onset of clinical symptoms, with potential for alleviation of clinical symptoms and possibly alleviation of the disease, includes “treatment” of the disease.

  "Needing treatment" includes mammals already having the disease or disorder, and mammals susceptible to the disease or disorder, and mammals to which the disease or disorder is to be prevented.

  In another embodiment, a therapeutically effective dose of a mutated TNFSF protein, mutated TNFSF gene, or mutated TNFSF antibody is administered to a patient having a disease that includes inappropriate expression of the TNFSF protein. A “disease involving inappropriate expression of TNFSF protein” within the scope of the present invention is a disease characterized by abnormal TNFSF protein due to a change in the amount of TNFSF protein or due to the presence of a TNFSF protein variant. Or the inclusion of obstacles. Excess includes any cause, including but not limited to overexpression at the molecular level, prolonged or accumulated appearance at the site of action, or enhanced activity of TNFSF protein compared to normal conditions. Can also be attributed. This definition includes diseases or abnormalities characterized by a decrease in TNFSF protein. This reduction includes, but is not limited to, decreased expression at the molecular level, shortened or reduced appearance at the site of action, mutations in the TNFSF protein, or decreased TNFSF protein activity relative to normal conditions. Not due to any cause. Such an excess or decrease in TNFSF protein compared to normal expression, appearance, or activity of the TNFSF protein can be measured according to, but not limited to, the assays described and cited herein.

Administration of the mutant TNFSF protein of the present invention, preferably in the form of a sterile aqueous solution, is oral, subcutaneous, intravenous, intranasal, intraaural, transdermal, topical (eg, gel, salve, lotion, cream, etc.) , Intraperitoneal, intramuscular, intrapulmonary (eg, AERx® inhalable technique sold by Aradigm or Inhance ^TM pulmonary delivery system sold by Inhale Therapeutics), intravaginal, intrarectal, or intraocular Can be performed in various ways, including but not limited to. In some examples, the mutant TNFSF protein may be applied directly as a solution or spray, for example, in the treatment of wounds, inflammation, and the like. Depending on the mode of introduction, the pharmaceutical composition can be formulated in various ways.

  Sustained release or controlled release formulations may also be used in the compositions of the present invention. For example, a microsphere-based delivery system composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG), ProLease® (sold by Alkermes), and Other pharmaceutically compatible polymerizable matrices can be used to create sustained release formulations.

  The in-formulation concentration of the therapeutically active mutant TNFSF protein can vary from about 0.1 to 100% by weight. In another preferred embodiment, the concentration of the mutant TNFSF protein ranges from 0.003 to 1.0 molar and is 0.03, 0.05, 0.1, 0.2, 0.0 per kilogram body weight. A dose of 3 mmol is preferred.

  The pharmaceutical composition of the present invention comprises a mutant TNFSF protein in a form suitable for administration to a patient. In a preferred embodiment, the pharmaceutical composition is in a water-soluble form, such as present as a pharmaceutically acceptable salt, which means that it includes both acid and base addition salts. “Pharmaceutically acceptable acid addition salts” include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and acetic acid, propionic acid, glycolic acid, pyruvic acid, succinic acid, maleic acid, malon Free base formed with organic acids such as acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid Represents a salt that retains its biological potency and is not biologically or otherwise harmful. “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as isopropylamine. , Salts of trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine.

  The pharmaceutical composition may include one or more of the following: carrier proteins such as serum albumin; buffers such as NaOAc; bulking agents such as microcrystalline cellulose, lactose, corn and other starches; binders; Sweeteners and other flavors; colorants; and polyethylene glycols. Additives are well known in the art and are used in various formulations.

In a further embodiment, the mutant TNFSF protein is added to a micelle formulation; see US Pat. No. 5,833,948, which is hereby incorporated by reference in its entirety.
A combination of pharmaceutical compositions may be administered. In addition, the composition may be administered in combination with other therapeutic agents.

  In certain embodiments provided herein, antibodies, including but not limited to monoclonal and polyclonal antibodies, are raised against mutant TNFSF proteins using methods known in the art. In a preferred embodiment, these anti-mutant TNFSF antibodies are used for immunotherapy. Accordingly, a method of immunotherapy is provided. “Immunotherapy” means the treatment of a TNFSF-related disease with an antibody raised against a mutant TNFSF protein. As used herein, immunotherapy can be passive or active. Passive immunotherapy as defined herein is the passive transfer of antibodies to a recipient (patient). Active immunization is the induction of antibody and / or T cell responses in a recipient (patient). Induction of the immune response may be the result of providing the recipient with a mutant TNFSF protein antigen against which antibodies are made. As will be appreciated by those skilled in the art, mutant TNFSF protein antigens can be obtained by injecting a recipient with a mutant TNFSF polypeptide against which an antibody is desired to be generated or by It may be provided by contacting a mutant TNFSF protein-encoding nucleic acid capable of expressing a TNFSF protein antigen under conditions for expression of the mutant TNFSF protein antigen.

  In another preferred embodiment, the therapeutic compound is conjugated to an antibody, preferably an anti-mutant TNFSF protein antibody. The therapeutic compound can be a cytotoxic agent. In this method, targeting a cytotoxic agent to tumor tissue or cells results in a reduction in the number of affected cells, thereby reducing symptoms associated with cancer and mutant TNFSF-related diseases. Cytotoxic substances are numerous and diverse and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, cursin, crotin, phenomycin, enomycin and the like. Cytotoxic substances are also radiochemicals created by conjugating radioisotopes to antibodies made against cell cycle proteins or by conjugating radionuclides to chelating agents covalently bound to antibodies. Is included.

  In a preferred embodiment, the mutant TNFSF protein can be administered as a therapeutic agent and formulated as outlined above. Similarly, mutant TNFSF genes (including full-length sequences, partial sequences, or regulatory sequences of mutant TNFSF coding regions) can be administered in gene therapy applications, as is known in the art. These mutant TNFSF genes can include antisense applications as gene therapy (ie, for integration into the genome) or as antisense compositions, as will be appreciated by those skilled in the art.

  In a preferred embodiment, a nucleic acid encoding a mutant TNFSF protein can also be used for gene therapy. In gene therapy applications, genes are introduced into cells to achieve in vivo synthesis of therapeutically effective gene products, eg, for replacement of defective genes. “Gene therapy” includes both conventional gene therapy, where a permanent effect is achieved by a single treatment, and administration of a gene therapy agent that includes a single or repeated administration of a therapeutically effective DNA or mRNA. Include. Antisense RNA and DNA can be used as therapeutic agents to block the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be drawn into cells and have low cellular concentrations due to limited uptake by the cell membrane, where they act as inhibitors [Zamecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146 (1986)]. The oligonucleotides can be modified to enhance their uptake, for example by replacing negatively charged phosphodiester groups with uncharged groups.

  There are a variety of techniques available for introducing nucleic acids into viable cells. This technique depends on whether the nucleic acid is transferred to cultured cells in vitro or to the intended host cell in vivo. Suitable techniques for transferring nucleic acids to mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection [Dzau et al., Trends in Biotechnology 11: 205-210. (1993)]. In some situations, it may be desirable to provide the nucleic acid source with a substance that targets the target cell, such as a cell surface membrane protein or antibody specific for the target cell, a ligand for a receptor on the target cell, and the like. When employing liposomes, proteins that bind to cell surface membrane proteins associated with endocytosis can be used for targeting and / or promoting uptake. For example, capsid proteins or fragments thereof directed to specific cell types, antibodies to proteins that undergo cyclic internalization, proteins that target intracellular localization and increase intracellular half-life. Receptor-mediated endocytosis techniques are described, for example, by Wu et al., J. Biol. Chem. 262: 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87: 3410- 3414 (1990). For a review of gene marking and gene therapy protocols, see Anderson et al., Science 256: 808-813 (1992).

  In other embodiments, the mutant TNFSF gene is administered as a DNA vaccine, either a single gene or a combination of mutant TNFSF genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16: 1304-1305 (1998). Methods of using genes as DNA vaccines are well known to those skilled in the art and include placing a mutant TNFSF gene or a portion of a mutant TNFSF gene under the control of a promoter for expression in a patient in need of treatment. To do.

  The mutant TNFSF gene used in the DNA vaccine may encode the full-length mutant TNFSF protein, but more preferably encodes a portion of the mutant TNFSF protein including a peptide derived from the mutant TNFSF protein. Yes. In a preferred embodiment, a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a mutant TNFSF gene. Similarly, it is possible to immunize a patient with multiple mutant TNFSF genes or portions thereof as defined herein. Without being bound by theory, expression of polypeptides encoded by the DNA vaccine, cytotoxic T cells, helper T cells and antibodies are induced, which recognize and destroy or eliminate cells expressing TNFSF protein.

  In a preferred embodiment, the DNA vaccine includes a gene encoding an adjuvant molecule with the DNA vaccine. Such adjuvant molecules include cytokines that increase the immunogenic response to the mutant TNFSF polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those skilled in the art and have use in the present invention.

All references cited herein, including patents, patent applications (provisional, utility models and PCT) and publications, are hereby incorporated by reference in their entirety.
The following examples serve to illustrate the mode of use of the invention in more detail and to describe the best mode contemplated for carrying out various aspects of the invention. It will be understood that these examples are not intended to limit the true scope of the invention in any way, but rather are presented for illustrative purposes.

Example
Expression, purification of TNF-alpha library and activity assay of TNF-alpha variants Methods:
1) Preparation of overnight cultures Transformability in 96 well PCR plates Tuner (DE3) pLyS cells were transformed with 1 μl of TNF-alpha library DNA, 34 mg / ml chloramphenicol and 100 mg / ml Spread on LB agar plates with ampicillin. After overnight growth at 37 ° C., colonies were picked from each plate in 1.5 ml CG medium with 34 mg / ml chloramphenicol and 100 mg / ml ampicillin contained in a 96 deep well block. The block was shaken overnight at 250 rpm and 37 ° C.

2) Expression Colonies were picked from the plate in 5 ml CG medium (34 mg / ml chloramphenicol and 100 mg / ml ampicillin) in a 24-well block and grown at 37 ° C., 250 rpm until OD600 0.6. At that time, IPTG was added to each well to a concentration of 1 mM. The culture was grown for an additional 4 hours.

3) Degradation The 24-well block was centrifuged at 3000 rpm for 10 minutes. The pellet was resuspended in 700 μl of degradation buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole). After freezing at −80 ° C. for 20 minutes and thawing at 37 ° C. (twice), MgCl ₂ was added to 10 mM and DNase I was added to 75 mg / ml. The mixture was incubated at 37 ° C. for 30 minutes.

4) Ni NTA column purification Purification was performed according to the Qiagen Ni NTA spin column purification protocol for non-denaturing conditions. The purified protein was dialyzed against 1 × PBS for 4 hours at 4 ° C. for 1 hour. Millipore multiscreen GV filter plates were used to filter sterilize the dialyzed protein to allow subsequent protein addition to the sterile mammalian cell culture.
5) Quantification The purified protein was quantified by SDS PAGE and subjected to Coomassie staining and Kodak (registered trademark) digital image density measurement.

TNF-alpha antagonist activity A) Materials and methods: Seeded cells for assay: seeded WEHI at 2.5 × 10 ⁵ cells / ml (50 μl / well). Assay medium was prepared as follows: 1X Assay Medium, 40 ml complete RPMI medium, 80 μl actinomycin D (final concentration 2 μg / ml). Assay medium + wild type TNF-alpha was prepared (wild type TNF-alpha is 1.1 mg / ml-1 μg / ml: 1: 1000; 1 ml of stock solution in 1 ml of RPMI—1 μg-20 ng / ml: 1 μg / ml : 50; 800 μl in 40 ml of assay medium TNF alpha variants were diluted as follows.

5) Dilution for inhibition assay:
Stock solution for diluting TNF receptor (TNF R) in 1X assay medium: Stock solution is 100 μg / ml. For 20 μg / ml: 1: 5 dilution: 60 μl of 100 μg / ml stock solution + 240 μl of 1 × assay medium with wild type TNF-alpha

As shown below, TNFR assay medium (finally 10 ng / ml on cells) containing 20 ng / ml wild-type TNF-alpha was diluted:

  All the above dilutions were made 16 hours prior to addition to the cells. Thereafter, 120 μl of each diluted sample was incubated at 4 ° C., and 120 μl of each sample was incubated at 37 ° C. The next morning, 50 μl of each sample was added to the cells. Cells were incubated for 4 hours at 37 ° C. After 4 hours of incubation, 100 μl of caspase substrate was added to each well followed by incubation at 37 ° C. for 2 hours. Read fluorescence. The results are shown in FIG.

TNF-alpha antagonist activity of combined TNF-alpha variants Materials and methods:
Cells were seeded for assay: WEHI164-13Var cells were seeded at 7.5 × 10 ⁵ cells / ml (50 μl / well) and incubated overnight at 37 ° C. to prepare assay medium (10 ×, final concentration on cells is 10 ng / mL) (complete RPMI 7 ml, 310 μg / mL wild type his-TNF [Lot # 263-56] 5 μL, 1 mg / mL actinomycin D 140 μL). TNF-alpha mutant dilutions were made by mixing these samples 3 days before the start of the experiment as follows:

  After all dilutions were made, 108.4 assay medium 68.4 (34.2 for TNF R) containing WThisTNFa was added to each dilution well. The 96 wells were then placed in the incubator for 3 days. 50ul of each sample was added to WEHI164-13Var cells for 4 hours. When the incubation was complete, 100 ul of caspase substrate was added. Incubated for 1.5 hours. An R110 curve was also prepared by diluting the R110 standard 1: 100 in RPMI and continuing with 8-point 1/2 dilution. 100 ul of each dilution was added to the plate without cells. These dilutions were made immediately before the substrate was added to the cells. 100 ul of substrate was also added to the R110 curve dilution. Once the 1.5 hour incubation at 37 ° C. was complete, all samples were read using a Wallac Fluorometer at 484/535 nm wavelength. The results are shown in FIG.

Phosphate buffered saline (PBS) with 0.01 mg / mL wild-type his-TNF [lot # 263-56] together with 0.1 mg / mL mutant TNF-alpha in 50 μL of the TNF-alpha mutant fixed equilibrium screening reaction solution A 1:10 fixed equilibrium ratio TNF-alpha variant was prepared by mixing in.

This mixture was prepared and incubated at 37 ° C. for 3-4 days. Cells were seeded for assay: Human U937 cells were seeded at 1 × 10 ⁶ cells / ml (50 μl / well) and incubated at 37 ° C. overnight.

Caspase assay Complete RPMI medium was warmed and 2 ug / mL actinomycin D was added. Each 50 uL reaction was mixed with 450 uL of RPMI medium supplemented with actinomycin D. This mixture was diluted 11 times 1: 1 to generate a fixed equilibrium dose curve. 50 uL of the diluted mixture was applied to the cells in quadruplicate. Cells were incubated for 1.5 hours in TNF-alpha / TNF-alpha mutant fixed equilibrium. When incubation was complete, 100 ul of caspase substrate was added and incubated for 1.5 hours. An R110 curve was also prepared by diluting the R110 standard 1: 100 in RPMI and continuing with 8-point 1/2 dilution. 100 ul of each dilution was added to the plate without cells. These dilutions were made immediately before the substrate was added to the cells. 100 ul of substrate was also added to the R110 curve dilution. Once the 1.5 hour incubation at 37 ° C. was complete, all samples were read using a Wallac Fluorometer at 484/535 nm wavelength. The results are shown in FIGS. 6A-C.

Binding Assay TNFa biotinylation was performed by adding 20 molar excess of Sulfo-NHS-LC-biotin to the protein sample and incubating the sample on ice for 2 hours. Excess biotin was removed from the sample by dialysis. The coupling rate was in the range of 1 to 4. The protein concentration of biotinylated TNFa was measured by BCA protein assay (Pierce). The wells of the microtiter plate were coated with an anti-FLAG antibody at a concentration of 2.5 mg / ml and blocked with 3% BSA overnight at 4 ° C. The FLAG-tagged protein TNFR1 receptor was added to the wells of an anti-FLAG coated microtiter plate at a concentration of 10 ng / ml in PBS + 1% BSA and the plate was incubated for 2 hours at room temperature. Biotinylated TNFa protein in a concentration range of 0-1 mg / mL was added in 4 parts to anti-FLAG-TNFR1-coated wells to represent total binding. Nonspecific binding was determined by adding 4 parts of biotinylated TNF alpha protein in a concentration range of 0-1 μg / ml to wells coated with anti-FLAG antibody alone. Binding occurred at + 4 ° C overnight to ensure equilibration. Alkaline phosphatase-conjugated neutravidin (Pierce) was added to the wells at a 1: 10,000 dilution in PBS + 1% BSA and incubated for 30 minutes at room temperature. CSPD star substrate (Applied Biosystems, Foster City, Calif.) Was added to detect and measure luminescence (Wallac VICTOR, Perkin Elmer Life Sciences, Boston, Mass.). Specific binding of TNFa was calculated by subtracting non-specific binding from total binding. The data was fitted to the binding equation y = (BLmax ^* x) / (Kd + x).
The results of the binding assay are shown in FIG. All variants showed reduced receptor binding.

Example 7
TNF-alpha mutants exchanged with wild-type TNF-alpha and tested TNF-alpha mutants that reduce NFkB activation are A145R, double mutant A145R / Y87H and triple mutant E146K / V91E / N34E. there were. His-tagged TNF-alpha was preincubated for 3 days at 37 ° C. with a 10-fold excess (1:10) of the various mutants. Wild-type TNF-alpha alone and pre-exchanged TNF-alpha mutant heterotrimers were tested for the ability to activate the NFkB agonist luciferase reporter (pNFkB-luc, Clontech) in 293T cells. 293T cells were seeded in 96-well plates at 1.2 × 10 ⁴ cells / well. Fugene transfection reagent is used according to the manufacturer's protocol (Roche), pNFkB-luc (NF-kB dependent luciferase reporter) or pTal (control: basal promoter that drives the luciferase gene but does not contain the NFkB binding element ) To transform the cells. Twelve hours after transfection, cells were treated with a final concentration of 10 ng / ml wild type TNF-alpha or a pre-exchanged mixture of 10 ng / ml: TNF / 100 ng / ml variant. After 12 hours of treatment, Steady-Glo Luciferase Assay System (Promega) was used according to the manufacturer's protocol and cells in 96-well plates were processed for luciferase assays. Luminescence from each well was measured using a Packard TopCount NXT (Packard Bioscience) luminescence counter. The treated samples were tested in 4 parts, and for each treatment, the mean value of luminescence was plotted as the value of the bar including standard deviation. The results are shown in FIG. 20A. The graph shows that the TNF-alpha variant of the present invention was effective in reducing NFkB activation induced by wild-type TNF-alpha. The TNF-alpha variant A145R / Y87H was most effective in reducing NFkB activation induced by TNF-alpha.

Immunolocalization of NFkB in HeLa cells HeLa cells were seeded on 12 mm sterile coverslips (Fisherbrand) at a density of 1.5 × 10 ⁵ cells / well in a 6-well plate at 37 ° C., 5% CO ₂ atmosphere. Cultured. The next day, various concentrations of his-tagged wild type TNF-alpha, A145R / Y87H mutant alone, or his-tagged TNF-alpha and a 10-fold excess of A145 / Y87H mutant (previously exchanged at 37 ° C. for 3 days. ) To treat the cells at 37 ° C., 5% CO ₂ . After 30 minutes incubation, cells attached to the coverslips in 6-well plates were briefly washed with PBS and fixed with 4% formaldehyde / PBS for 10 minutes. Cells were then washed 5 more times with PBS or kept overnight in the final PBS wash before processing the cells for immunocytochemistry. Cells fixed on coverslips were treated with 0.1% Triton X-100 / PBS. Buffer was aspirated and cells on the coverslip were blocked at 37 ° C. for 15 minutes in a humidified chamber with 50 ul of 0.1% BSA / 0.1% TX-100 / PBS per coverslip. The blocking reagent was removed and replaced with a primary antibody against the p65 subunit of NF-kB (pAb C-20, Santa Cruz Bioscience). After 1 hour incubation at 37 ° C., the antibody was removed and the coverslip was washed 5 times with PBS. 50 ul of FITC-conjugated secondary antibody (Jackson Immunolaboratories) diluted in block buffer (1: 100) was added to each coverslip (Jackson Immunolaboratories), and the coverslips were incubated for an additional hour in a light-tight humidified chamber. The next antibody was removed by 5 washes with PBS. Cover slips were rinsed briefly with distilled water, air dried in a light-tight chamber, and mounted on slides using Anti-fade (Molecular Probes). Using a Nikon Eclipse TS100 microscope combined with a Cool SNAP-Pro CCD camera (Media Cybernetics) to capture digital images of antibody-reactive cells using FITC filters and 40x objective lens, and using Image Pro Plus software (Media Cybernetics) And operated.

  FIG. 4B shows a photograph of the immunolocalization of NFkB in HeLa cells. This indicates that the exchange of wild-type TNF-alpha and A145 / Y87H TNF-alpha mutants inhibits TNF-alpha-induced nuclear translocation of NFkB in HeLa cells. TNF-alpha variant A145R / Y87H alone does not induce NFkB nuclear translocation, unlike wild-type TNF-alpha. In addition, wild-type TNF-alpha exchanged to form a heterotrimer (10 days excess of TNF-alpha mutant A145R / Y87H) (3 days, 37 ° C.) Loss the ability to induce nuclear translocation. This data is consistent with the effect of this variant in the luciferase reporter assay.

TNFA variant A145R / Y87H reduces His-tagged wild-type TNF-alpha, TNF-alpha variant A145 / Y87H and exchanged wild-type TNF to reduce TNF-alpha-induced activation of the NFkB agonist luciferase reporter -Alpha: A145R / Y87H heterotrimer (10-fold excess of TNF-alpha variant A145R / Y87H exchanged at 37 ° C for 1 day) was tested in the NFkB luciferase reporter assay as in Example 7A above. . The experiment showed a wider range of final TNF-alpha concentrations and increasing doses (0.78, 1.56, 3.13, 6.25, 12.5, 25 ng / ml) at each TNF-alpha concentration of 10 The procedure was as in Example 7A, except that it was used with a fold excess of TNF-alpha variant (A145R / Y87H).

  Wild-type TNF-alpha: A145R / Y87H heterotrimer significantly reduced activation levels. This shows the inhibitory effect of the TNF-alpha A145R / Y87H variant on wild type TNF-alpha. As the lower dotted line in FIG. 4C shows, unlike wild-type TNF-alpha, TNF-alpha variant A145 / Y87H alone has no significant agonizing effect on NFkB activation. As the gray solid line in FIG. 4C shows, reporters without NFkB binding elements do not respond to TNF-alpha, so activation induced by wild-type TNF-alpha is NFkB activation dependent.

Antagonistic RANKL mutant library design Antagonistic RANKL mutants were generated using a dominant negative strategy involving single strand diversity and by selection of competitive inhibitory antagonists.

A dominant-negative RANKL library variant PDA ^™ was used to generate a human structural model of RANKL and select amino acids that disrupt RANK binding. Since there was no crystal structure of human OPGL, it was necessary to create a homology model. Here, PDATM was used for side chain substitution based on the human sequence and mouse RANKL structure (PDB # 1JTZ). This model formation was facilitated by 87% sequence identity between the mouse and human RANKL sequences. The dominant negative RANKL treatment strategy is based on the design of a novel RANKL variant with reduced receptor binding and / or activation properties and the ability to form heterotrimers with wild-type RANKL (FIG. 21). In other words, a RANKL variant that does not activate RANK exchanges with the wild-type RANKL protein, sequesters it into an inactive heterotrimer, and inhibits its activity. This treatment results in a decrease in osteoclast number and activity, leading to decreased bone resorption and increased overall bone mineral density.

A dominant negative RANKL variant was designed by replacing the amino acid at the key RANKL-RANK contact point with an amino acid that disrupts the receptor's ability to activate the receptor. The PDA ^™ technique was used to select substitutions suitable for optimal protein folding. Exchange of these trimeric RANKL variants with trimeric wild type RANKL results in inactivation of wild type RANKL and reduced osteoclast formation. To more effectively help achieve this goal, the RANKL variant can also be designed to preferentially heterotrimerize with wild-type RANKL.

Single Chain Dominant Negative Polypeptides There are a number of strategies for covalently linking monomers. This consists of a polypeptide linkage between the N and C termini of two domains consisting of zero or more amino acids (resulting in a single chain polypeptide containing multiple domains); via a disulfide bond between monomers Linkage; includes but is not limited to linkage via a chemical crosslinker.

  A number of strategies exist to modify individual domains such that receptor binding is removed (or reduced). This includes amino acid modifications that create steric reciprocity between the ligand domain and the receptor; amino acid modifications that create electrostatic reciprocity; modifications that create desolvation of adverse amino acids; and ligand / receptor interface Including, but not limited to, chemical modification of amino acids at (eg, PEGylation or glycosylation).

Linking RANKL monomers to trimers To improve the dominant negative behavior of these mutants, a single chain dimer is created between two modified receptor interaction domains.

Construction of RANKL mutant library Full-length RANKL cDNA was cloned into lymph node cDNA using RT-PCR. After PCR-mediated addition of TEV and HIS tags, two lengths of RANKL, amino acids 147-317 and 159-317 were subcloned into pET33b.

  A mutagenesis reaction was performed to introduce nucleotide changes leading to the desired amino acid substitution. Mutagenesis reactions utilize branched and overlapping pairs of oligonucleotides with nucleotide variations that introduce specific amino acid changes. These oligonucleotides are extended through multiple thermal cycles using Pfu polymerase and the products are transfected into TOP10 bacterial cells after cutting these extension products with DpnI. The clones were sequenced to confirm the designed changes and to ensure that no modifications were introduced into the cDNA during thermal cycling.

RANKL variant screening for non-agonists, superagonists, and antagonists A library of 89 human RANKL variants was constructed. All members of the library contain the amino acid sequence of FIG. 1b and two solubility-imparting substitutions, C221S / I247E, and 89 inhibitory modifications designed by computer processing. These 89 human RANKL mutant proteins were expressed and purified in E. coli and 52 were screened for non-agonists, super-agonists, and antagonists in an assay that monitors osteoclast development in RAW 264.7 cells. RAW264.7 cells were treated with purified human RANKL mutant protein and non-agonists and superagonists were identified by measuring released TRAP. Of the 52 RANKL variants screened, 21 RANKL variants were identified as non-agonists, while C221S / I247E / A172R was found to be a superagonist. In addition, the above antagonism screening identified six antagonism variants. These antagonistic variants have the ability to reduce the osteoclastogenic activity of human RANKL.

Bioassay for Tartrate Resistant Acid Phosphatase (TRAP) Activity A bioassay for TRAP activity was used to assay non-agonist, superagonist and antagonism. After adding RANKL mutant protein to cell lines such as RAW264.7, TRAP activity was measured to identify RANKL mutants that are non-agonists or superagonists (FIGS. 23-25, 29). To identify RANKL mutants of antagonists, the RANKL mutant protein is mixed with one of the active RANKL soluble mutants, C221S / I247E, the protein mixture is added to RAW264.7, and TRAP activity is measured. Further screening was performed. An antagonist RANKL variant that reduces the activity of the RANKL soluble variant C221S / I247E was identified.

RAW264.7 cells (ATCC # TIB-71) were incubated in a 75 cm ² flask in a 5% CO ₂ humidified incubator at 37 ° C. with 2 mM L-glutamine (Gibco-BRL # 12571-063), 10% heat inactivated. Cultured and maintained in growth medium containing a-MEM containing 100 units / ml and 100 μg / ml of fetal bovine serum (FBS) (Hyclone # SH30071.03) and penicillin / streptomycin (Gibco-BRL # 15140-122), respectively. .

  Recombinant mouse RANKL (R & D # 462-TR), recombinant human soluble RANKL (Biosource # PHP0034), and recombinant human soluble RANKL mutant were combined with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone # SH30071. 03) and penicillin / streptomycin (Gibco-BRL # 15140-122) in an assay medium consisting of a-MEM containing l-glutamine (Gibco-BRL # 12571-063) supplemented with 100 units / ml and 100 μg / ml respectively. Diluted to its working concentration. RANKL standards and 250 ml of sample were serially diluted 1: 2 by adding triplicate or 4 parts to a 96-well assay plate and transferring 125 ml to the next well containing 125 ml of assay medium.

The exhausted medium was aspirated, 10 ml of pre-warmed medium was added to the flask, and the cells were collected by scraping the cells from the flask using a cell scraper. Cells were transferred to a 15 ml conical tube, spun at 1000 rpm for 5 minutes and resuspended in 10 ml assay medium. Cells were counted in 96-well assay plates, were seeded at a density of 1.5 × 10 ^3/125 ml / well. The assay plate was incubated for 72 hours in a 37 ° C., 5% CO ₂ humidified incubator.

  The amount of osteoclast-like cells induced from RAW264.7 was determined by measuring tartrate resistant acid phosphatase (TRAP) activity. TRAP substrate was added by adding 20 mg of p-nitrophenol phosphate (pNPP) (ICN # 100878) to 10 ml of TRAP buffer pH 5.0 (100 mM sodium acetate, 11.5 mg / ml sodium tartrate (ICN # 195503)). Prepared. The medium was aspirated from the assay plate and discarded. Cells were fixed with 1: 1 ethanol and acetone solution for 1 minute and washed once with PBS (Hyclone # SH30256.02) 100-150 ml / well. 100 ml of TRAP solution was added to each well and the plate was incubated at 37 ° C. for 2 hours. The reaction was stopped by adding 50 ml / well of 0.2N NaOH solution and the absorbance was read at 405 nm. When the absorbance reached saturation, the solution was diluted 1: 5 and read again.

Cytological staining of tartrate-resistant acid phosphatase (TRAP) In parallel experiments, osteoclast development from RAW264.7 cells was used, with cytological staining due to the presence of tartrate-resistant acid phosphatase (TRAP) multinuclear positive cells And measured. Prior to staining, 0.1 M acetate buffer was prepared by combining 35.2 ml of 0.2 M sodium acetate solution, 14.8 ml of 0.2 M acetic acid solution and 50 ml of water. TRAP buffer pH 5.0 (50 mM acetate buffer, 30 mM sodium tartrate (Sigma # S-8640), 0.1 mg / ml Napthol AS-MX Phosphate Disodium Salt (Sigma # N-5000), 0.1% Triton X- 100) 10 ml was prepared fresh for each assay. TRAP buffer was warmed in a 37 ° C. water bath, 0.3 mg / ml Fast Red Violet LB Stain (Sigma # F-3381) was added, and the stain was returned to the 37 ° C. water bath. The medium was aspirated from the assay plate and discarded. Cells were washed once with 150 ml / well of PBS (Hyclone # SH30256.02) and then fixed with 100 ml / well of 10% glutaraldehyde solution for 15 minutes at 37 ° C. Cells were washed twice with pre-warmed PBS 150 ml / well. 100 ml of pre-warmed TRAP stain was added to each well and the plate was incubated at 37 ° C. for 5-10 minutes. TRAP stain was removed and 100 ml PBS was added to each well to prevent the cells from drying out. TRAP positive multinucleated cells (3 or more nuclei) were counted. The results are shown in FIG.

Other TNFSF variants BlyS, CD40L, APRIL, OX-40 were generated according to the protocol disclosed above. They were exchanged for the corresponding wild type TNFSF members. APRIL and BlyS were exchanged between them and the corresponding APRIL or BlyS. Binding assays and dose response curves were performed for each TNFSF member.

  While particular embodiments of the present invention have been described by way of illustration, those skilled in the art will recognize that many modifications may be made in the details without departing from the invention as set forth in the appended claims. Is done.

FIG. 1 shows the overall mechanism by which a dominant negative TNFSF protein can thereby antagonize the action of a naturally produced TNFSF protein. FIG. 2 shows the structural overlap of TNFSF ligand monomers. FIG. 3 shows the multiple sequence alignment (MSA) of the human TNFSF member group. FIG. 4A is a chart showing that the TNF-alpha variant group has been pre-exchanged with wild-type TNF-alpha to reduce TNF-alpha-induced NFkB activation in 293T cells. FIG. 4B shows NFkB immunity in HeLa cells, showing that replacement of wild-type TNF-alpha with A145 / Y87HTNFSF mutant inhibits TNFSF-induced nuclear translocation of NFkB in HeLa cells. Target-a location specific photo. FIG. 4C shows that TNFSF variant A145R / Y87H reduces wild type TNFSF-induced activation of the NFkB-induced luciferase reporter. FIG. 5 is a chart showing the antagonist activity of the TNF-alpha variant. 6A-C are dose response curves for caspase activation by various TNF-alpha variants. FIG. 7A shows a dominant-negative inhibitory RANKL mutant strategy model. FIG. 7B shows a list of computer designed inhibitory RANKL variants. FIG. 7C shows that RANKL soluble mutant C22S / I247E-mediated osteoclast development is antagonized by OPG and RANK-Fc. FIG. 7D shows the RANKL mutant antagonist screen. FIG. 7E shows a RANKL mutant antagonism screen using a fixed ratio of RANKL-C221S / I247E to a 10-fold excess mutant. FIG. 7F shows the RANKL mutant antagonist screen. FIG. 7G shows a summary of RANKL antagonistic variants.

Claims (44)

  A method for antagonizing a naturally occurring TNFSF protein, wherein the naturally occurring TNFSF monomer protein is contacted with a mutant TNFSF monomer comprising at least one mutant extracellular domain of the TNFSF protein, and a mixed TNFSF oligomer Forming.   The method of claim 1, wherein the mixed oligomers have reduced receptor signaling compared to wild type oligomers.   The method of claim 2, wherein the mixed oligomer is substantially incapable of activating receptor signaling.   The method of claim 1, wherein the mixed oligomer interacts with a receptor interface at at least one receptor binding site such that the receptor is substantially incapable of activating receptor signaling.   The mutant TNFSF monomer protein comprises at least one receptor contact domain having reduced affinity for the desired receptor relative to its corresponding wild type TNFSF protein, and other TNFSF monomers The method of claim 1, wherein the method retains the ability to interact with.   2. The method of claim 1, wherein the mutant TNFSF monomeric protein physically interacts with a naturally occurring TNFSF monomeric protein and reduces the ability of the naturally produced TNFSF to activate at least one receptor. .   The method according to claim 1, wherein the mutant TNFSF protein is a fusion protein.   The method according to claim 1, wherein the mutant protein is chemically modified.   9. The method of claim 8, wherein the chemical modification is PEGylation.   The method of claim 8, wherein the mixed oligomer comprises covalent cross-linkage between monomers.   The method of claim 10, wherein the mixed oligomer comprises at least one linker peptide between monomers.   12. The method of claim 11, wherein the linker peptide is a sequence of at least one and no more than about 30 amino acid residues.   13. The method of claim 12, wherein the linker peptide is a sequence of at least 5 and not more than about 20 amino acid residues.   14. The method of claim 13, wherein the linker peptide is a sequence of at least 10 and no more than about 15 amino acid residues.   The method of claim 11, wherein the linker peptide comprises at least one of Gly, Ser, Ala, or Thr.   A method for making a mixed TNFSF oligomer comprising: mutating TNFSF protein comprising at least one mutated extracellular domain of TNFSF monomer protein; homo-oligomer comprising naturally produced TNFSF monomer protein; and at least one naturally produced product A method comprising contacting TNFSF monomer under conditions that exchange a mutated monomer with a mutant monomer to form a mixed oligomer.   A mixed TNFSF oligomer comprising at least one mutated TNFSF protein comprising at least one mutated extracellular domain of a TNFSF monomer protein, and a naturally produced TNFSF monomer protein.   A mutant TNFSF monomer protein comprising at least one mutant extracellular domain of a TNFSF protein.   A mutant TNFSF protein, wherein the mutant TNFSF protein interacts with a receptor interface at at least one receptor binding site, preventing the receptor from substantially activating receptor signaling.   Contains at least one receptor contact domain with reduced affinity for the desired receptor relative to its corresponding wild type TNFSF protein and retains the ability to interact with other receptor interaction domains 19. The mutant TNFSF protein according to claim 18.   19. A mixed TNFSF oligomer comprising at least one mutant TNFSF protein monomer according to claim 18, wherein said mixed oligomer has a reduced ability to activate the corresponding receptor relative to a wild-type oligomer. Mixed TNFSF oligomer.   19. The mutant TNFSF protein of claim 18, wherein the mutant TNFSF protein physically interacts with a naturally produced TNFSF protein to form a mixed trimer.   19. A mixed TNFSF oligomer comprising at least one mutant TNFSF protein monomer according to claim 18 comprising a modification at a receptor contact position.   19. The mutant TNFSF monomer protein according to claim 18, wherein the mutant TNFSF protein includes a modification at a trimer intermediate surface position.   19. The mutant TNFSF protein of claim 18, wherein said mutant TNFSF protein physically interacts with its corresponding naturally occurring TNFSF protein.   19. The mutant TNFSF protein of claim 18, wherein said mutant TNFSF protein physically interacts with a non-corresponding naturally produced TNFSF protein.   A recombinant nucleic acid encoding the mutant TNFSF monomer protein according to any one of claims 18 to 26.   An expression vector comprising the recombinant nucleic acid according to claim 27.   A host cell comprising the recombinant nucleic acid of claim 27.   A host cell comprising the expression vector according to claim 28.   A method for producing a non-naturally occurring TNFSF protein, comprising culturing the host cell according to claim 29 or claim 30 under conditions suitable for expression of the nucleic acid.   32. The method of claim 31, further comprising recovering the TNFSF protein.   27. A pharmaceutical composition comprising the mutant TNFSF protein according to any one of claims 18 to 26 and a pharmaceutically acceptable carrier.   27. A method for treating a TNFSF-related disorder, comprising administering the mutant TNFSF protein according to any one of claims 18 to 26 to a patient in need of treatment.   35. The method of claim 34, wherein the TNFSF-related disorder is an autoimmune disease.   19. The mutant TNFSF protein of claim 18, wherein at least one modification is non-conservative.   19. The mutant TNFSF protein of claim 18, wherein at least one modification is a surface modification.   37. The mutant TNFSF protein of claim 36, wherein the modification is located in a domain selected from the group consisting of a large domain, a small domain, a DE loop, a trimer interface and combinations thereof.   At least one of the large domain positions is a TNFA corresponding position 28, 29, 30, 31, 32, 33, 34, 63, 64, 65, 66, 77, 68, 69, 112, 113, 114, 115, 137, 40. The mutant TNFSF protein of claim 38, selected from the group consisting of 138, 139, 140, 141, 142, 143, 144, 145, 146 and 147.   40. The variant TNFSF of claim 38, wherein the at least one small domain position is selected from the group consisting of TNFA corresponding positions 72, 73, 74, 75, 76, 78, 79, 95, 96, 97 and 98. protein.   39. The mutant TNFSF protein of claim 38, wherein at least one of said DE loop positions is selected from the group consisting of TNFA corresponding positions 84, 85, 86, 87, 88 and 89.   At least one trimer intermediate surface position is a TNFA corresponding position 11, 13, 15, 34, 36, 53, 54, 55, 57, 59, 61, 63, 72, 73, 75, 77, 119, 87. 91, 92, 93, 94, 95, 96, 97, 98, 99, 102, 103, 104, 109, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 39. The mutant TNFSF protein of claim 38, selected from the group consisting of:, 124, 125, 147, 148, 149, 151, 155, 156 and 157.   43. The mutant TNFSF protein of claim 42, wherein the at least one trimer interface position is selected from the group consisting of 57, 34 and 91.   19. The mutant TNFSF protein of claim 18, wherein said mutant TNFSF protein antagonizes a soluble naturally produced TNFSF protein.

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